Pharmacological targeting of mRNA cap formation

ABSTRACT

This invention provides methods for the discovery of molecules that target an essential aspect of eukaryotic gene expression--the formation of the mRNA 5&#39; cap m7GpppN. An underlying principle of this invention is the use of a different strains of a test organism that differ only in the composition or source of the essential cap-forming enzymes. The invention provides isogenic yeast strains that derive all their capping activities from fungal sources versus mammalian sources. These strains form the basis of a differential growth inhibition assay to identify molecules that specifically target the fungal capping apparatus. This invention also provides a method to screen in vitro for molecules that inhibit fungal RNA triphosphatase, an essential enzyme that catalyzes the first of three steps in cap synthesis.

FEDERAL FUNDING LEGEND

This invention was created using federal funds under Grant No. GM-52470from the National Institutes of Health. Accordingly, the United StatesGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of genebiochemical pharmacology and drug discovery. More specifically, thepresent invention relates to method of screening for a compound thatinhibits formation of an organism's 5' mRNA cap structure.

2. Description of the Related Art

Processing of eukaryotic mRNA in vivo is coordinated temporally andphysically with transcription. The earliest event is the modification ofthe 5' terminus of the nascent transcript to form the cap structurem7GpppN. The cap is formed by three enzymatic reactions: (i) the 5'triphosphate end of the nascent RNA is hydrolyzed to a diphosphate byRNA 5' triphosphatase; (ii) the diphosphate end is capped with GMP byGTP:RNA guanylyltransferase; and (iii) the GpppN cap is methylated byAdoMet:RNA (guanine-N7) methyltransferase [1].

RNA capping is essential for cell growth. Mutations of thetriphosphatase, guanylyltransferase, or methyltransferase components ofthe yeast capping apparatus that abrogate catalytic activity are lethalin vivo [2-12]. Genetic and biochemical experiments highlight roles forthe cap in protecting mRNA from untimely degradation by cellular 5'exonucleases [13] and in recruiting the mRNA to the ribosome duringtranslation initiation [14].

The physical and functional organizations of the capping apparatusdiffer in significant respects in fungi, metazoans, protozoa, andviruses. Hence, the cap-forming enzymes are potential targets forantifungal, antiviral, and antiprotozoal drugs that would interfere withcapping of pathogen mRNAs, but spare the mammalian host capping enzymes.A plausible strategy for drug discovery is to identify compounds thatblock cell growth contingent on pathogen-encoded capping activitieswithout affecting the growth of otherwise identical cells bearing thecapping enzymes of the host organism. For this approach to be feasible,the capping systems of interest must be interchangeable in vivo.

The architecture of the capping apparatus differs between metazoans,fungi, protozoa, and DNA viruses. Metazoan species encode atwo-component capping system consisting of a bifunctionaltriphosphatase-guanylyltransferase polypeptide (named Mce1p in the mouseand Hce1p in humans) and a separate methyltransferase polypeptide (Hcm1pin humans) [6, 9, 15-22]. The budding yeast Saccharomyces cerevisiaeencodes a three component system consisting of separate triphosphatase(Cet1p), guanylyltransferase (Ceg1p), and methyltransferase (Abd1p) geneproducts [7, 10, 11, 23]. In yeast, the triphosphatase (Cet1p) andguanylyltransferase (Ceg1p) polypeptides interact to form a heterodimer[11], whereas in mammals, autonomous triphosphatase andguanylyltransferase domains are linked in cis within a singlepolypeptide (Mce1p) [18].

Vaccinia virus capping enzyme is a multifunctional protein thatcatalyzes all three reactions. The triphosphatase, guanylyltransferase,and methyltransferase active sites are arranged in a modular fashionwithin a single polypeptide--the vaccinia D1 protein [24-30]. Other DNAviruses encode a subset of the capping activities; e.g., baculovirusesencode a bifunctional triphosphatase-guanylyltransferase (LEF-4) andChlorella virus PBCV-1 encodes a monofunctional guanylyltransferase[31-33]. The guanylyltransferase and methyltransferase domains areconserved between DNA viruses, fungi, and metazoans. In contrast, thetriphosphatase components are structurally and mechanisticallydivergent.

RNA Guanylyltransferase--Transfer of GMP from GTP to the 5' diphosphateterminus of RNA occurs in a two-step reaction involving a covalentenzyme-GMP intermediate [34]. Both steps require a divalent cationcofactor, either magenesium or manganese.

    E+pppG←→E-pG+PPi                               (i)

    E-pG+ppRNA←→GpppRNA+E                          (ii)

The GMP is covalently linked to the enzyme through a phosphoamide (P--N)bond to the ε-amino group of a lysine residue within a conserved KxDGelement (motif I) found in all known cellular and DNA virus-encodedcapping enzymes (FIG. 1). Five other sequence motifs (III, IIIa, IV, V,and VI) are conserved in the same order and with similar spacing in thecapping enzymes from fungi, metazoans, DNA viruses, and trypanosomes(FIG. 1) [35].

Hakansson et al. [36] have determined the crystal structure of theChlorella virus capping enzyme in the GTP-bound state and with GMP boundcovalently. The protein consist of a larger N-terminal domain (domain 1,containing motifs I, III, IIIa, and IV) and a smaller C-terminal domain(domain 2, containing motif VI) with a deep cleft between them. Motif Vbridges the two domains. Motifs I, III, IlIa, IV, and V form thenucleotide binding pocket. The crystal structure reveals a largeconformational change in the GTP-bound enzyme, from an "open" to a"closed" state, that brings motif VI into contact with the β and γphosphates of GTP and reorients the phosphates for in-line attack by themotif I lysine. When the crystal is soaked in manganese,guanylyltransferase reaction chemistry occurs in crystallo and thecovalent enzyme-GMP intermediate is formed. However, only the enzyme inthe closed conformation is reactive.

Identification of essential enzymic functional groups has beenaccomplished by site-directed mutagenesis of Ceg1p, the RNAguanylyltransferase of Saccharomyces cerevisiae. The guanylyltransferaseactivity of Ceg1p is essential for cell viability. Hence, mutationaleffects on Ceg1p function in vivo can be evaluated by simple exchange ofmutant CEG1 alleles for the wild type gene. The effects of alaninesubstitutions for individual amino acids in motifs I, III, IIIa, IV, V,and VI [2, 5, 6] have been examined. Sixteen residues were defined asessential (denoted by asterisks in FIG. 1) and structure-activityrelationships at these positions were subsequently determined byconservative replacements. Nine of the essential Ceg1p side chainscorrespond to moieties which, in the Chlorella virus capping enzymecrystal structure, make direct contact with GTP (arrowheads in FIG. 2).These include: the motif I lysine nucleophile which contacts theα-phosphate of GTP; the motif I arginine and motif III glutamate, whichcontact the 3' and 2' ribose hydroxyls, respectively; the motif IIIphenylalanine, which stacks on the guanine base; the two motif Vlysines, which contact the α-phosphate; the motif V aspartate, whichinteracts with the β-phosphate; the motif VI arginine that interactswith the β phosphate; and the motif VI lysine, which contacts the γphosphate of GTP [6, 36].

On the basis of sequence conservation outside motifs I, III, lIIa, IV,V, and VI, the capping enzymes of fungi (S. cerevisiae, S. pombe, C.albicans,), metazoans (C. elegans and mammals) and Chlorella virus canbe grouped into a discrete subfamily [6, 37]. The sequence alignment inFIG. 2 highlights two motifs that are present in these capping enzymes,but not in the poxvirus enzymes, which can be designated motif P andmotif Vc. In the Chlorella virus capping enzyme, motif P forms one wallof the guanosine binding pocket of domain 1 [36]. MotifVc--(K/R)I(I/V)EC--is situated between motifs V and VI in domain 2. Theglutamate residue of motif Vc is essential for the activity of thefungal guanylyltransferase Ceg1p [37].

RNA Triphosphatase--There are at least two mechanistically andstructurally distinct classes of RNA 5' triphosphatases: (i) thedivalent cation-dependent RNA triphosphatase/NTPase family (exemplifiedby yeast Cet1p, baculovirus LEF-4, and vaccinia D1), which require threeconserved collinear motifs (A, B, and C) for activity [12, 28, 31, 32],and (ii) the divalent cation-independent RNA triphosphatases, e.g., themetazoan cellular enzymes and the baculovirus enzyme BVP [15, 17,38-40], which require the HCxAGxGR(S/T)G phosphate-binding motif. Theexistence of additional classes of RNA 5'-triphosphatases is likely,given that the candidate capping enzymes of several RNA viruses andtrypanosomatid protozoa lack the defining motifs of the two known RNAtriphosphatase families [41, 42]. Hence, the triphosphatase componentsof the capping apparatus provide attractive targets for theidentification of specific antifungal, antiviral, and antiprotozoaldrugs that will block capping of pathogen mRNAs, but spare the mammalianhost enzyme.

Mammalian RNA Triphosphatase--Metazoan capping enzymes consist of anN-terminal RNA triphosphatase domain and a C-terminalguanylyltransferase domain. In the 496-amino acid mouse enzyme Mce1p,the two catalytic domains are autonomous and nonoverlapping [18]. Themetazoan RNA triphosphatase domains contain a (I/V)HCxAGxGR(S/T)Gsignature motif initially described for the protein tyrosinephosphatase/dual-specificity protein phosphatase enzyme family. Theseenzymes catalyze phosphoryl transfer from a protein phosphomonoestersubstrate to the thiolate of the cysteine of the signature motif to forma covalent phosphocysteine intermediate, which is then hydrolyzed toliberate phosphate (FIG. 3). The metazoan capping enzymes hydrolyze thephosphoanhydride bond between the β and γ phosphates oftriphosphate-terminated RNA; they are not active on nucleosidetriphosphates. The conserved cysteine of the signature motif isessential for RNA triphosphatase function [11, 15, 38, 39]. Acharacteristic of the cysteine-phosphatases is their lack of arequirement for a divalent cation cofactor.

The N-terminal portion of Mce1p from residues 1-210 is an autonomous RNAtriphosphatase domain [18]. Recombinant Mce1(1-210)p has been expressedin bacteria and purified to near-homogeneity. Mce1(1-210)p sediments ina glycerol gradient as a discrete peak of 2.5 S, indicating that thedomain is monomeric in solution. The RNA triphosphatase activity ofMce1(1-210)p can be assayed by the release of ³² Pi from γ³² P-labeledpoly(A). A kinetic analysis showed that the initial rate of Pi releasewas proportional to enzyme concentration. Mce1(1-210)p hydrolyzed 1.2 to2 molecules of Pi per enzyme per second at steady state. RNAtriphosphatase activity was optimal in 50 mM Tris buffer at pH 7.0 to7.5. Activity was optimal in the absence of a divalent cation and wasunaffected by EDTA. Inclusion of divalent cations elicited aconcentration dependent inhibition of RNA triphosphatase activity. 75%inhibition was observed at 0.5 mM MgCl₂ or MnCl₂. Mce1(1-210) did notcatalyze release of ³² Pi from [γ³² P]ATP.

Metal-dependent RNA Triphosphatases--The RNA triphosphatases of S.cerevisiae and DNA viruses are structurally and mechanisticallyunrelated to the metazoan RNA triphosphatases. The vaccinia virus RNAtriphosphatase depends absolutely on a divalent cation cofactor.Vaccinia triphosphatase displays broad specificity in its ability tohydrolyze the γ phosphate of ribonucleoside triphosphates,deoxynucleoside triphosphates, and triphosphate-terminated RNAs [43,44]. The NTPase and RNA triphosphatase reactions occur at a singleactive site within an 545-amino acid N-terminal domain of vacciniacapping enzyme that is distinct from the guanylyltransferase active site[27-30]. The vaccinia RNA triphosphatase is optimal with magnesium, is12% as active in manganese, and is inactive with cobalt [43]. Incontrast, the vaccinia NTPase is fully active with cobalt, manganese, ormagnesium [43, 44]. Baculovirus LEF-4 hydrolyzes the γ phosphate of RNAand NTPs; the LEF-4 NTPase is activated by manganese or cobalt, but notby magnesium [31].

The yeast RNA 5'-triphosphatase Cet1p also hydrolyzes the γ phosphate ofnucleoside triphosphates [12]. The NTPase of Cet1p is activated bymanganese and cobalt. This is a property shared with the triphosphatasecomponents of the vaccinia D1 and baculovirus LEF-4 capping enzymes.Recent studies illuminate a common structural basis for metal-dependentcatalysis by these enzymes. The metal-dependent RNA triphosphatasesshare 3 collinear sequence motifs, designated A, B, and C (FIG. 4).These are present in yeast Cet1p, in the Cet1p homolog from yeastCandida albicans, in the triphosphatase-guanylyltransferase domains ofthe vaccinia virus, Shope fibroma virus, molluscum contagiosum virus,and African swine fever virus capping enzymes, and in baculovirus LEF-4.Mutational analysis identified several residues within these motifs thatare essential for the RNA triphosphatase and ATPase activities ofvaccinia virus capping enzyme; the essential residues include twoglutamates in motif A, an arginine in motif B, and two glutamates inmotif C [28]. Alanine substitutions at any of these positions in thevaccinia capping enzyme reduced phosphohydrolase specific activity by 2to 3 orders of magnitude. These 5 residues may comprise part of thetriphosphatase active site. All five residues essential for vacciniatriphosphatase activity are conserved in LEF-4 and Cet1p. Mutations ofthe equivalent residues of Cet1p result in loss of triphosphataseactivity [12].

Physical Association of the Triphosphatase and GuanylyltransferaseComponents of the Capping Apparatus--Yeast and mammals use differentstrategies to assemble a bifunctional enzyme with triphosphatase andguanylyltransferase activities. In yeast, separate triphosphatase(Cet1p) and guanylyltransferase (Ceg1p) enzymes interact to form aheterodimer, whereas in mammals, autonomous triphosphatase andguanylyltransferase domains are linked in cis within a singlepolypeptide (Mce1p).

Cet1p and Ceg1p Form a Heterodimeric Capping Enzyme Complex InVitro.--The native size of purified recombinant yeast RNA triphosphataseCet1p was gauged by glycerol gradient sedimentation. Cet1p sedimented asa single monomeric component of 4.3 S. The yeast guanylyltransferaseCeg1p also sediments as a monomer in a glycerol gradient. Yet, whenequal amounts of recombinant Cet1p and Ceg1p were mixed in buffercontaining 0.1 M NaCl and the mixture was analyzed by glycerol gradientsedimentation, the two proteins, as well as the triphosphatase andguanylyltransferase activities, cosedimented as a single discrete peakof 7.5 S. Thus, Ceg1p and Cet1p interact in vitro to form a heterodimer[11]. Cet1p does not heterodimerize with the structurally homologous RNAguanylyltransferase domain of the mouse capping enzyme. Recombinantmouse guanylyltransferase, Mce1(211-597)p, was purified to homogeneity,mixed with Cet1p or with a buffer control, and then subjected tosedimentation analysis in parallel with the Cet1-Ceg1 mixtures. The 45kDa Mce1(211-597) protein alone sedimented as a single monomeric peak[18]. Sedimentation of the Mce1(211-597)p plus Cet1p mixture revealed noshift in the distribution of the mouse guanylyltransferase or the yeasttriphosphatase to a more rapidly sedimenting form [11]. Hence, yeast RNAtriphosphatase forms a heterodimer with yeast guanylyltransferase, butnot with the mammalian enzyme. Subsequent studies of the Candidaalbicans RNA triphosphatase (named CaCet1p) showed that it couldinteract with S. cerevisiae guanylyltransferase Ceg1p in vivo as gaugedby a two-hybrid reporter assay [45].

Cet1p-Ceg1p Heterodimerization is Essential In Vivo--Truncated proteinsCet1(201-549)p and Cet1(246-549)p were expressed in bacteria andpurified from soluble bacterial lysates by Ni-agarose andphosphocellulose column chromatography. Purified Cet1(201-549)p andCet1(246-549)p catalyzed the release of ³² Pi from γ³² P-labeledtriphosphate-terminated poly(A) or [γ³² P]ATP with the same specificactivity as full-length Cet1p [11]. The CET1(201-259) gene in singlecopy was functional in vivo in supporting yeast cell growth [11]. Thefinding that the CET1(246-549) gene on a CEN plasmid could not supportcell growth, even though the gene product has full RNA triphosphataseactivity in vitro, suggests that the catalytic activity of Cet1p, thoughessential for cell growth (see below), may not suffice for Cet1pfunction in vivo. Glycerol gradient analysis showed that Cet1(201-549)pby itself sedimented as a monomer. When Cet1(201-549)p was mixed withCeg1p and the mixture was analyzed by glycerol gradient sedimentation,the two proteins cosedimented as a 6.8 S heterodimer [11]. The moreextensively truncated Cet1(246-549)p did not sediment as a discretemonomer like full-sized Cet1p and Cet1(201-549)p. Rather, most of theCet1(246-549)p sedimented as a high molecular weight oligomer (˜13 S)that retained RNA triphosphatase activity. When a mixture ofCet1(246-549)p and Ceg1p was sedimented most of the Cet1(246-549)premained aggregated; only a minor fraction of the input Ceg1p wasshifted to the size expected for a heterodimer. Deletion from residues201-245 (which results in loss of function in vivo despite retention oftriphosphatase activity in vitro) affects the interaction of Cet1p withCeg1p.

The implication of these data is that the interaction of Cet1p withCeg1p is essential for yeast cell growth. Pharmacological interferencewith Ceg1p-Cet1p heterodimerization is a potential mechanism forblocking gene expression in fungi without impacting on mammalian cells.

Cap Methyltransferase--The enzyme RNA (guanine-N7-)methyltransferase(referred to hereafter as cap methyltransferase) catalyzes the transferof a methyl group from AdoMet to the GpppN terminus of RNA to producem7GpppN-terminated RNA and AdoHcy [1]. The Saccharomyces cerevisiae capmethyltransferase is the product of the ABD1 gene [7]. ABD1 encodes a436-amino acid polypeptide. A catalytic domain of Abd1p from residues110 to 426 suffices for yeast cell growth (8, 9); this segment of Abd1pis homologous to the methyltransferase catalytic domain of the vacciniavirus capping enzyme [7]. A key distinction between the yeast andvaccinia virus cap methyltransferases is their physical linkage, or lackthereof, to the other cap-forming enzymes. The vaccinia virusmethyltransferase active site is encoded within the same polypeptide asthe triphosphatase and guanylyltransferase, whereas the yeastmethyltransferase is a monomeric protein that is not associated with theother capping activities during fractionation of yeast extracts [7].Mutational analyses of the yeast and vaccinia cap methyltransferaseshave identified conserved residues that are critical for cap methylation[8, 9, 46]. In the case of Abd1p, mutations that abolishedmethyltransferase activity in vitro were lethal in vivo.

A putative cap methyltransferase from C. elegans was identified onphylogenetic grounds [9]. An alignment of the sequence of the predicted402-amino acid C. elegans protein (Genbank accession Z81038) with theyeast cap methyltransferase Abd1p is shown in FIG. 5. Although itremains to be demonstrated that the nematode protein has capmethyltransferase activity, the extensive sequence conservationsuggested that other metazoans might also encode homologues of Abd1p. Ahuman cDNA that encodes a bona fide cap methyltransferase has beenidentified and a physical and biochemical characterization of therecombinant human cap methyltransferase (Hcm1p) produced in bacteria wasconducted. A functional C-terminal catalytic domain of Hcm1p was definedby deletion analysis [22].

Interaction of the Cellular Capping Apparatus with the PhosphorylatedCTD of RNA Polymerase II. Cap formation in eukaryotic cells in vivo istargeted to the nascent chains synthesized by RNA polymerase II (potII). A solution to the problem of how pot II transcripts arespecifically singled out for capping has been described whereby thecellular capping enzymes are targeted to pre-mRNA by binding to thephosphorylated carboxyl-terminal domain (CTD) of the largest subunit ofpol II [16-18, 47, 48]. The CTD, which is unique to pol II, consists ofa tandem array of a heptapeptide repeat with the consensus sequenceTyr-Ser-Pro-Thr-Ser-Pro-Ser. The mammalian pol II large subunit has 52tandem repeats whereas the S. cerevisiae subunit has 27 copies. The polII largest subunit exists in two forms, a nonphosphorylated IIA form anda phosphorylated IIO form, which are interconvertible and functionallydistinct. In vivo, the pol IIO enzyme contains as many as 50phosphorylated amino acids (primarily phosphoserine) within the CTD.During transcription initiation, pol IIA is recruited to the DNAtemplate by the general transcription factors. The pol IIA CTD undergoesextensive phosphorylation and conversion to IIO during the transitionfrom preinitiation complex to stable elongation complex. Several CTDkinase activities have been implicated in CTD hyperphosphorylation, eachof which contains a cyclin and cyclin-dependent kinase subunit pair. Thecdk7 and cyclin H subunits of the general transcription factor TFIIHcatalyze phosphorylation of Ser-5 of the CTD heptapeptide. Other CTDkinases include the cdk8/cyclin C pair found in the pol II holoenzyme,CTDK-I, a heterotrimeric kinase with cdk-like and cyclin-like subunits,and P-TEFb, a regulator of polymerase elongation with a cdc2-likesubunit.

The recombinant S. cerevisiae and Sc. pombe guanylyltransferases Ceg1pand Pce1p bind specifically to the phosphorylated form of the CTD [16].Moreover, recombinant yeast cap methyltransferase Abd1p also bindsspecifically to CTD-PO4 [16]. Phosphorylation at Ser-5 of the heptadrepeat was sufficient to confer guanylyltransferase andmethyltransferase binding capacity to the CTD [16]. This analysis hasbeen extended to mammalian capping enzyme where the key finding is thatthe guanylyltransferase domain Mce1(211-597)p by itself binds toCTD-PO4, whereas the triphosphatase domain Mce1(1-210)p does not [18].These findings suggest that the mammalian RNA triphosphatase is targetedto the nascent pre-mRNA by virtue of its connection in cis to theguanylyltransferase. The phosphorylation-dependent interaction betweenguanylyltransferase and the CTD is conserved from yeast to mammals. Itis not clear if the structural elements on the yeast and mammalianenzymes that interact with CTD-PO4 are conserved or divergent.Nonetheless, pharmacological interference with the binding ofguanylyltransferase or cap methyltransferase to the CTD is a potentialmechanism for blocking gene expression in fungi or mammalian cells. Suchinterference can occur either by direct blocking of cappingenzyme/CTD-PO4 binding or indirectly by affecting the phosphorylationstate of the CTD.

The prior art is deficient in the lack of methods of screening for acompound that inhibits formation of an organism's 5' mRNA cap structure.The present invention fulfills this longstanding need in the art.

SUMMARY OF THE INVENTION

The present invention facilitates the discovery of drugs that target anessential aspect of eukaryotic gene expression--the formation of themRNA 5' cap m7GpppN. The underlying principle of the invention is theuse of a different strains of a test organism that differ only in thecomposition or source of the essential cap-forming enzymes. For example,the construction of isogenic yeast strains that derive all their cappingactivities from fungal sources versus mammalian sources provides thebasis to identify molecules that specifically target the fungal cappingapparatus.

The methods disclosed herein test a battery of candidate molecules fortheir ability to selectively impair the growth of a strains containingcapping enzymes differing in composition or source. In a simpleembodiment of the invention, this would entail the local application ofan array of candidate molecules to agar culture plates that had beeninoculated with an engineered yeast strain containing capping enzymesdiffering in composition or source. The plates are incubated to permitgrowth of the yeast cells to form a confluent lawn. Growth inhibition bythe applied compound is detected as a "halo" of no-growth or slow-growtharound the site of application. The methods will necessarily detect manycompounds that inhibit the growth of all test strains; these areregarded as non-specific to capping and would likely not be pursued. Thepositive candidates are those that inhibit growth of one strain, but notof an otherwise identical strain containing a capping apparatus thatdiffers in composition or source. For example, molecules that inhibitthe growth of yeast cells containing an all-fungal capping apparatus,but have little or no effect on the growth of yeast cells containing themammalian capping apparatus, would be regarded as promising leads forantifungal drugs. Conversely, molecules that inhibit the growth of cellscontaining an all-mammalian capping apparatus, but have little or noeffect on the growth of cells containing the fungal capping apparatusmerit further consideration as specific inhibitors of capping inmammalian cells, with the potential for development as an antineoplasticagent.

The methods of the present invention are also applicable to theidentification of potential antiviral agents and antiparasitic agentsthat specifically target virus-encoded or parasite-encoded cappingenzymes. In this embodiment of the invention, the growth inhibitionscreen would include testing the array of candidate molecules against astrain that contains one or more virus-encoded or parasite-encodedcapping activities in lieu of the endogenous enzyme(s). Molecules thatselectively inhibit growth of the strain bearing the viral or parasitecapping enzyme component, but not both of the strain bearing themammalian capping apparatus, would be regarded as promising leads forfurther evaluation of antiviral or antiparasitic activity.

The invention is not restricted to the use of fungi as the testorganisms. Advances in gene targeting in mammalian cells make itfeasible to construct mammalian cell lines in which one or more of thegenes encoding cellular capping activities is deleted and replaced by agene encoding the analogous enzyme from another source. Thus, candidatecap-targeting compounds could be identified by screening in parallel forselective growth inhibition of one of several cell lines differing onlyin the composition or source of the capping apparatus. Eukaryoticviruses that depend on virus-encoded capping activities can also bedeveloped as the targets for testing of growth inhibition. In thisembodiment, a viral gene encoding an essential capping activity would bedeleted and replaced by a gene encoding the analogous enzyme fromanother source. Virus plaque formation on permissive host cells providesan easy visual screen (by plaque number and plaque size) for inhibitionof virus replication by candidate agents added to the medium. Agentsthat selectively inhibit the replication of virus containing cappingenzymes from one source, but not from another source, are presumed to doso by selective targeting of cap formation.

The invention is also not restricted to identifying exogenous moleculesthat target cap formation. Another embodiment facilitates the DNA-basedidentification of natural or synthetic gene products that inhibit cellgrowth via intracellular effects on the capping enzymes. In thisapplication of the invention, candidate genes or gene libraries (eithernatural or synthetic) would be transformed into a test yeast strain,e.g., a strain bearing an all-fungal capping apparatus. The genes in thelibrary are under the control of a regulated promoter (e.g. a GALpromoter) so that their expression can be repressed (in glucose medium)or induced (in galactose medium) by the experimenter. The initial screenselects for library-transformed cells that grow on glucose, but areinhibited on galactose. Plasmids recovered from such cells would beclonally amplified in bacteria and then re-transformed in parallel intoyeast strains containing the fungal capping apparatus and strainscontaining one or both mammalian capping enzymes. Plasmids that elicitgalactose-dependent growth inhibition of the strain with the fungalcapping system, but do not inhibit the strains with mammalian cappingcomponents, are good candidates to encode specific antagonists of fungalcap formation. Sequencing the plasmid encoded gene product will revealthe identity of the presumptive inhibitor. Structure-activityrelationships for the gene product can then be delineated by DNA-basedmutagenesis.

The growth-inhibiting molecules or genes identified using the methodsdescribed in this invention could conceivably target cap formation via anumber of distinct mechanisms, including: (i) direct inhibition of thecatalytic activity of one of the cap-forming enzymes by the identifiedmolecule or a metabolite thereof; (ii) interference with protein-proteininteractions required for in vivo function of one of the cap-formingenzymes; (iii) alterations in the level of available substrates for capformation (e.g., GTP and AdoMet) or the level of endogenous inhibitorsof cap formation (e.g., AdoHcy); and (iv) the synthesis of abnormal capstructures in the presence of the growth-inhibiting molecule (ormetabolites thereof) that effectively "poison" cellular transactionsdependent on the RNA cap.

An advantage of the present invention is that it is geared to detectspecific targeting of capping enzymes in vivo based on differences intheir composition or origin, without bias with respect to the mechanismof inhibition. Once candidate molecules are identified, further testingof growth inhibition of isogenic strains differing in one component ofthe capping system can reveal which enzyme is targeted. Furthermechanistic studies can ensue using purified capping enzymes from therelevant sources.

The invention also encompasses an in vitro screen to identify candidateinhibitors of the catalytic activity of the fungal RNA 5'triphosphatase. The method exploits the fact that the yeast RNAtriphosphatase Cet1p has a vigorous ATPase activity that depends oneither manganese or cobalt as the divalent cation cofactor [12]. Themethod is simple, quantitative, and adaptable to a calorimetricdetection assay that is suited to high-throughput screening forinhibitors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the guanylyltransferase signature motifs. Six collinearsequence elements, designated motifs I, III, IIIa IV, V, and VI, arepresent in cellular and viral capping enzymes. The amino acid sequencesare aligned for the enzymes of S. cerevisiae (Sce), S. pombe (Spo), C.albicans (Cal), Chlorella virus PBCV-1 (ChV), mouse (Mus), African swinefever virus (ASF), Trypanosoma brucei gambiense (Tbr), Crithidiafasciculata (Cfa), AcNPV baculovirus (Lef4), vaccinia virus (Vac), Shopefibroma virus (SFV), and molluscum contagiosum virus (MCV). The numbersof amino acid residues separating the motifs are indicated. The aminoacids of the yeast enzyme motifs that are essential for Ceg1p functionin vivo are denoted by asterisks.

FIG. 2 shows the conserved sequence elements in fungal, metazoan, andChlorella virus mRNA capping enzymes. The amino acid sequence of the S.cerevisiae (sce) guanylyltransferase Ceg1p from residues 43 to 399 isaligned with the homologous regions of the guanylyltransferases encodedby Candida albicans (cal), Schizosaccharomyces pombe (spo), mouse (mus),Caenorhabditis elegans (cel), and Chlorella virus PBCV-1. Eightconserved collinear sequence elements, designated motifs P, I, III, IIIaIV, V, Vc, and VI, are shown in shaded boxes. Gaps in the sequence areindicated by dashes (-). Ceg1p residues defined by mutational analysisas essential for enzyme function in vivo are denoted by asterisks.Residues in proximity to GTP in the Chlorella virus capping enzyme-GTPco-crystal are indicated by arrowheads below the aligned sequences.

FIG. 3 shows the RNA triphosphatase domains of metazoan capping enzymesand baculovirus phosphatase BVP. The amino acid sequence of the168-amino acid baculovirus phosphatase BVP (Baculo) is aligned with theN-terminal RNA triphosphatase domains of mouse capping enzyme (Mus CE)and C. elegans capping enzyme (Cel CE). Gaps in the sequences areindicated by dashes (-). Amino acids conserved in all three proteins aredenoted by asterisks. The protein phosphatase signature motif ishighlighted in the shaded box. The active site cysteine is in bold face.The presumptive reaction pathway involving formation of a cysteinylphosphate intermediate is shown.

FIG. 4 shows the conserved sequence elements of the metal-dependent RNAtriphosphatases. Three conserved motifs, designated A, B, and C, in theRNA triphosphatases of S. cerevisiae (Cet1), vaccinia virus (vvD1),Shope fibroma virus (SFV), molluscum contagiosum virus (MCV), Africanswine fever virus (ASF), baculovirus (Lef4), and Candida albicans (Cal)are aligned in the figure. Also included in the alignment is thepredicted translation product of the S. cerevisiae YMR180c open readingframe (180c). Cet1p residues conserved in at least two other familymembers are shaded. The numbers of amino acids separating the motifs areindicated. The five amino acids in the vaccinia virus capping enzymethat are essential for triphosphatase activity are underlined. Cet1presidues Glu-305, Glu-307, Arg-454, Glu-492, Glu-494, and Glu-496 thatare essential for CET1 function in vivo and for Cet1p RNA triphosphateactivity in vitro are denoted by arrowheads. The locations of motifs A,B, and C within the yeast 456-amino acid RNA triphosphatase Cet1p isdiagrammed above the aligned sequences. The margins of the C-terminalcatalytic domain sufficient for γ-phosphate hydrolysis are indicated bybrackets.

FIG. 5 shows the amino acid sequence conservation in human and yeast capmethyltransferases. The complete amino acid sequence of the 476-aminoacid Hcm1p protein (hcm) is aligned with the complete sequence of yeastAbd1p (abd) and with the predicted 402-amino acid C. elegans C25A1.fgene product (cel) from residue 1 to 373. Gaps in the sequences areindicated by dashes (-). The C-termini of Hcm1p and Abd1p are indicatedby asterisks. Residues in Hcm1p that are identical or similar in Abd1por the C. elegans protein are shown in shaded boxes. Amino acidsessential for Abd1p function are denoted by dots.

FIG. 6 panels A and B show the mutational effects on the RNAtriphosphatase activity of Cet1(201-549)p. (A) Protein purification.Aliquots (3 μg) of the Ni-agarose preparations of recombinant wild type(WT) Cet1(201-549)p and the indicated Cet1(201-549)p-Ala mutants wereelectrophoresed through a 12% polyacrylamide gel containing 0.1% SDS.Polypeptides were visualized by staining with Coomassie blue dye. Thepositions and sizes (in kDa) of marker proteins are indicated on theleft. (B) RNA triphosphatase activity. Reaction mixtures (10 μl)containing 50 mM Tris HCl (pH 7.5), 5 mM DTT, 1 mM MgCl₂, 20 pmol (oftriphosphate termini) of [γ³² P]-poly(A), and either WT or mutantproteins as specified were incubated for 15 min at 30° C. Aliquots ofthe mixtures were applied to a polyethyleneimine-cellulose TLC plate,which was developed with 0.75 M potassium phosphate (pH 4.3). ³² Pirelease is plotted as a function of input protein.

FIG. 7 shows the hydrolysis of ATP by Cet1p. Reaction mixtures (10 μl)containing 50 mM Tris HCl (pH 7.5), 5 mM DTT, 1 mM [γ³² P]ATP, 250 ng ofrecombinant Cet1p (phosphocellulose fraction), and 1 mM divalent cationas specified were incubated for 15 min at 30° C. An aliquot (4 μl) ofthe mixture was applied to a polyethyleneimine-cellulose TLC plate,which was developed with 0.5 M LiCl₂, 1 M formic acid. An autoradiographof the TLC plate is shown. The positions of Pi and ATP are indicated onthe left.

FIG. 8 panels A-D, show a kinetic analysis of ATP hydrolysis by Cet1p.(A) Protein titration. Reaction mixtures (10 μl) containing 50 mM TrisHCl (pH 7.0), 5 mM DTT, 2 mM MnCl₂, 1 mM [γ³² P]ATP, and either Cet1p,Cet1(201-549)p, or Cet1(246-549)p were incubated for 15 min at 30° C. Pirelease is plotted as a function of input protein. (B) Kinetics.Reaction mixtures (100 μl) containing 50 mM Tris HCl (pH 7.0), 5 mM DTT,2 mM MnCl₂, 1 mM [γ³² P]ATP, and either 8, 16, or 32 nM Cet1p wereincubated at 30° C. Aliquots (10 μl) were withdrawn at the timesindicated and quenched immediately by the addition of 2.5 μl of 5 Mformic acid. Pi release is plotted as a function of time. (C) Hydrolysisof [α³² P]ATP. Reaction mixtures (100 μl) containing 50 mM Tris HCl (pH7.0), 5 mM DTT, 2 mM MnCl₂, 1 mM [α³² P]ATP, and 100 ng of Cet1p wereincubated at 30° C. Aliquots (10 μl) were withdrawn at the timesindicated and quenched with formic acid. The products were analyzed byTLC. The levels of [α³² P]ADP (o) and [α³² P]AMP (q) are plotted as afunction of time. An otherwise identical reaction containing 1 mM [γ³²P]ATP was analyzed in parallel; Pi release (.). (D) ATP-dependence.Reaction mixtures (20 μl) containing 50 mM Tris HCl (pH 7.0), 5 mM DTT,2 mM MnCl₂, 40 pg of Cet1p, and [γ³² P]ATP as indicated were incubatedfor 15 min at 30° C. Pi release is plotted as a function of ATPconcentration. Inset: a double-reciprocal plot of the data is shown.

FIG. 9 shows cap methyltransferase activity of recombinant Hcm1p. Thecomplete reaction mixture (lane 1) contained (in 10 μl) 50 mM Tris HCl(pH 7.5), 5 mM DTT, 16 fmol of cap-labeled poly(A), 50 μM AdoMet, and˜50 fmol of Hcm1p (Ni-agarose 0.2 M imidazole eluate fraction). Reactioncomponents were varied as follows: omit AdoMet (lane 2); omit AdoMet,include 50 μM AdoHcy (lane 3); include 50 μM AdoMet plus 500 μM AdoHcy(lane 4); omit Hcm1p (lane 5); omit Hcm1p, include purified recombinantvaccinia capping enzyme (lane 6). After incubation at 37° C. for 10 min,the reaction mixtures were heated at 95° C. for 5 min, then adjusted to50 mM sodium acetate (pH 5.5). The samples were incubated with 5 μg ofnuclease P1 for 30-60 min at 37° C. The digests were then spotted onpolyethyleneimine cellulose TLC plates that were developed with 0.2 M(NH₄)₂ SO₄. An autoradiograph of the chromatogram is shown. Thechromatographic origin and the positions of cap dinucleotides m7GpppAand GpppA are indicated on the right.

FIG. 10 panels A-C show glycerol gradient sedimentation of human capmethyltransferase. The Ni-agarose fractions of Hcm1p (panel A), andN-terminal deletion mutants Δ120 (panel B) and Δ151 (panel C) weresedimented in glycerol gradients as described under Methods. Fractionswere collected from the bottom of the tubes (fraction 1). Aliquots (20μl) of alternate fractions were analyzed by SDS-PAGE along with analiquot of the material that had been applied to the gradient (lane Ni).The gels were fixed and stained with Coomassie blue dye. The positionsand sizes (kDa) of coelectrophoresed marker polypeptides are indicatedat the left of each gel. Methyltransferase reaction mixtures contained16 fmol of cap-labeled poly(A), 50 μM AdoMet, and 1 μl of a 1/500dilution of the indicated fractions from the Hcm1p and Δ120 glycerolgradients or 1 μl of a 1/50 dilution of fractions from the Δ151 glycerolgradient. The reaction products were digested with nuclease P1 andanalyzed by TLC. The extent of cap methylation [m7GpppA/(m7GpppA+GpppA)]was determined by scanning the chromatogram using a FUJIX BAS1000phosphorimager. The peaks of marker proteins catalase, BSA andcytochrome C, which were centrifuged in a parallel gradient, areindicated by arrowheads.

FIG. 11 panels A and B, show Cap methylation by N-terminal deletionmutants of Hcm1p. (A) Polypeptide composition. Aliquots (10 μg) of thepeak glycerol gradient fractions of wild type Hcm1p and deletion mutantsΔ120 and Δ151 were analyzed by SDS-PAGE. Polypeptides were visualized bystaining with Coomassie Blue dye. The positions and sizes (kDa) ofmarker polypeptides are indicated at the left. (B) Methyltransferaseactivity. Reaction mixtures contained 20 fmol of cap-labeled poly(A), 50μM AdoMet, and WT, Δ120, or Δ151 enzyme as specified.

FIG. 12 panels A and B, show Human cap methyltransferase substitutes foryeast Abd1p in vivo. Yeast strain YBS10 (MATα ura3 trp1 lys2 his3 leu2abd1::hisG [pCEN URA3 ABD1]) was transformed with 2μ TRP1 plasmids(panel A) or CEN TRP1 plasmids (panel B) containing the wild type HCM1gene or N-terminal deletion mutants as specified. Controltransformations were performed using the TRP1 vectors alone and a CENTRP1 plasmid containing ABD1. Trp+ isolates were streaked on agar platescontaining 0.75 mg/ml 5-FOA. The plates were photographed afterincubation for 3 days at 30° C.

FIG. 13 panels A and B show replacement of the yeast capping apparatuswith mammalian capping enzymes. (A) Yeast strain YBS50 (Δcet1 Δceg1) wastransformed with CEN TRP1 plasmids containing the indicated genes. Trp+isolates were streaked on agar containing 0.75 mg/ml 5-FOA. The platewas photographed after incubation for 3 days at 30° C. (B) Isogenicstrains deleted at one or more yeast capping enzyme loci weretransformed with CEN TRP1 plasmids as specified below and then selectedon 5-FOA. FOA-resistant isolates were streaked on a YPD agar plate. Theplate was photographed after incubation for 3 days at 30° C. HCM1Δ120MCE1 is YBS52 transformed with HCM1(121-476) and MCE1; ABD1 CET1 CEG1 isYBS52 transformed with ABD1 CET1 and CEG1 on a single plasmid; ABD1 MCE1is YBS50 transformed with MCE1; HCM1Δ120 CET1 CEG1 is YBS40 transformedwith HCM1(121-476).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of screening for acompound that inhibits formation of an organism's 5' mRNA cap structure,comprising the steps of: a) replacing a host organism's genes encoding5' mRNA capping functions with genes encoding 5' mRNA capping functionsfrom a first organism, thereby producing a host organism expressing thefirst organism's capping apparatus; b) replacing a host organism's genesencoding 5' mRNA capping functions with genes encoding the 5' mRNAcapping functions from a second organism, thereby producing a hostorganism expressing the second organism's capping apparatus, whereinsaid first organism and said second organism are not the same; c)treating the host organism expressing the first organism's cappingapparatus and the host organism expressing the second organism's cappingapparatus with a test compound; and d) comparing growth inhibition ofthe host organism expressing the first organism's capping apparatus withgrowth inhibition of the host organism expressing the second organism'scapping apparatus, wherein growth inhibition of only the host organismexpressing the first organism's capping apparatus relative to the hostorganism expressing the second organism's capping apparatus indicatesthat the test compound inhibits the 5' mRNA capping functions of thefirst organism, whereas growth inhibition of only the host organismexpressing the second organism's capping apparatus relative to the hostorganism expressing the first organism's capping apparatus indicatesthat the test compound inhibits the 5' mRNA capping functions of thesecond organism. Representative host organisms are selected from thegroup consisting of viruses, fungal cells, insect cells, plant cells,and mammalian cells. When the host organism is a virus, comparison ofgrowth inhibition of the virus expressing the first organism's cappingapparatus with the virus expressing the second organism's cappingapparatus is determined by the number of viral plaques formed onviral-permissive host cells and the viral plaque size formed onviral-permissive host cells. A preferred fungal host organism isSaccharomyces cerevisiae. Preferably, the first organism and secondorganism are selected from the group consisting of viruses, fungi,protozoa, plants, insects and mammals. Generally, the test compound willbe either a chemical, a drug or protein. Preferably, the protein isencoded by DNA that can be expressed in the host organism, wherein theDNA is operably linked to an inducible promoter. Preferably, the 5' mRNAcapping function inhibited by this method is RNA triphosphatase, RNAguanylyltransferase, or RNA (guanine-N7)-methyltransferase.

The present invention is directed to a method of screening for acompound that inhibits formation of an organism's 5' mRNA cap structure,comprising the steps of: a) replacing Saccharomyces cerevisiae's genesencoding 5' mRNA capping functions with genes encoding 5' mRNA cappingfunctions from a first organism, thereby producing S. cerevisiae cellsexpressing the first organism's capping apparatus; b) replacing S.cerevisiae's genes encoding 5' mRNA capping functions with genesencoding the 5' mRNA capping functions from a second organism, therebyproducing S. cerevisiae cells expressing the second organism's cappingapparatus, wherein said first organism and said second organism are notthe same; c) treating S. cerevisiae cells expressing the firstorganism's capping apparatus and S. cerevisiae cells expressing thesecond organism's capping apparatus with a test compound; and d)comparing growth inhibition of the S. cerevisiae cells expressing thefirst organism's capping apparatus with growth inhibition of the S.cerevisiae cells expressing the second organism's capping apparatus,wherein growth inhibition of only S. cerevisiae cells expressing thefirst organism's capping apparatus relative to S. cerevisiae cellsexpressing the second organism's capping apparatus indicates that thetest compound inhibits the 5' mRNA capping functions of the firstorganism, whereas growth inhibition of only S. cerevisiae cellsexpressing the second organism's capping apparatus relative to S.cerevisiae cells expressing the first organism's capping apparatusindicates that the test compound inhibits the 5' mRNA capping functionsof the second organism. Preferably, the first organism and secondorganism are selected from the group consisting of viruses, fungi,protozoa, plants, insects and mammals. Generally, the test compound willbe either a chemical, a drug or protein. Preferably, the protein isencoded by a DNA expressed in the S. cerevisiae cells, wherein the DNAis operably linked to an inducible promoter. Preferably, the 5' mRNAcapping function inhibited by this method is RNA triphosphatase, RNAguanylyltransferase, or RNA (guanine-N7)-methyltransferase.

The present invention is directed to a method of screening for acompound that inhibits formation of an organism's 5' mRNA cap structure,comprising the steps of: a) replacing Saccharomyces cerevisiae's genesencoding 5' mRNA capping functions with genes encoding 5' mRNA cappingfunctions from a fungal organism, thereby producing S. cerevisiae cellsexpressing the fungal organism's capping apparatus; b) replacing S.cerevisiae's genes encoding 5' mRNA capping functions with genesencoding the 5' mRNA capping functions from a mammalian organism,thereby producing S. cerevisiae cells expressing the mammalianorganism's capping apparatus; c) treating S. cerevisiae cells expressingthe fungal organism's capping apparatus and S. cerevisiae cellsexpressing the mammalian organism's capping apparatus with a testcompound; and d) comparing growth inhibition of the S. cerevisiae cellsexpressing the fungal organism's capping apparatus with growthinhibition of the S. cerevisiae cells expressing the mammalianorganism's capping apparatus, wherein growth inhibition of only S.cerevisiae cells expressing the fungal organism's capping apparatusrelative to S. cerevisiae cells expressing the mammalian organism'scapping apparatus indicates that the test compound inhibits the 5' mRNAcapping functions of the fungal organism, whereas growth inhibition ofonly S. cerevisiae cells expressing the mammalian organism's cappingapparatus relative to S. cerevisiae cells expressing the fungalorganism's capping apparatus indicates that the test compound inhibitsthe 5' mRNA capping functions of the mammalian organism. Preferably, the5' mRNA capping function inhibited by this method is RNA triphosphatase,RNA guanylyltransferase, or RNA (guanine-N7)-methyltransferase.

The present invention is directed to a method of screening for acompound that inhibits the catalytic activity of fungal RNA 5'triphosphatase, comprising the steps of: a) contacting said fungal RNA5' triphosphatase with a nucleoside triphosphate and a divalent cationcofactor in the presence and absence of a test compound; and detectinghydrolysis of said nucleoside triphosphate, wherein a lack of hydrolysisof said nucleoside triphosphate or a reduction in the hydrolysis of saidnucleoside triphosphate indicates inhibition of said fungal RNA 5'triphosphatase by said test compound. Preferably, the divalent cationcofactor is selected from the group consisting of manganese and cobalt.Although detection of hydrolysis may be by any method readily known tothose having ordinary skill in this art, preferable methods includeradioisotope assay and a calorimetric assay.

The present invention is also directed to a transformed host organism,wherein said host organism's genes encoding 5' mRNA capping functionsare replaced with replacement genes encoding 5' mRNA capping functions.Representative host organisms which can be transformed as describedbelow include viruses, fungal cells, insect cells, plant cells, andmammalian cells. Preferably, replacement genes are from an organismselected from the group consisting of viruses, fungi, protozoa, plants,insects and mammals. A preferred host organism is Saccharomycescerevisiae, and a preferred strain of Saccharomyces cerevisiae is YBS52(Δcet1 Δceg1 Δabd1). Even more preferably, the replacement genes arefrom an organism selected from the group consisting of a fungus and amammal, wherein replacement genes from the fungus are ABD1, CET1 andCEG1, and the resultant genotype is MATa leu2 ade2 trp1 his3 ura3 can1ceg1::hisG cet1::LEU2 abd1::KAN p360-CET1/CEG1/ABD1, or wherein thereplacement genes from said mammal are HCM1 and MCE1.

EXAMPLE 1

Expression and Purification of Yeast RNA Triphosphatase

Induced expression of the His-tagged Cet1p, Cet1(201-549)p, andCet1(246-549)p in Escherichia coli BL21(DE3) cells was performed asdescribed by Ho et al. [11]. The recombinant proteins were purified fromsoluble bacterial lysate by Ni-agarose and phosphocellulose columnchromatography [11].

EXAMPLE 2

Mutational Analysis of Yeast RNA Triphosphatase

Alanine substitution mutations were introduced into the CET1(201-549)gene by PCR using the two-stage overlap extension method. Residuestargeted for amino acid substitutions were Glu-305, Glu-307, Arg-454,Glu-492, Glu-494, and Glu-496. Plasmid p358-CES5(201-549) was thetemplate for the first stage amplifications. The DNA products of thesecond stage amplification were digested with NdeI and BamH1 andinserted into pET-16b. The presence of the desired mutations wasconfirmed by DNA sequencing; the inserted restriction fragment wassequenced completely in order to exclude acquisition of unwantedmutations during amplification and cloning. The His-tagged mutantproteins and the wild type protein were purified from soluble bacteriallysates by Ni-agarose chromatography as described by Ho et al. [11]. The0.2 M imidazole eluate fractions containing Cet1(201-549)p were dialyzedagainst buffer C (50 mM Tris HCl [pH 8.0], 50 mM NaCl, 2 mM DTT, 10%glycerol, 0.05% Triton X-100). Protein concentration was determined byusing the Bio-Rad dye reagent with bovine serum albumin as the standard.

EXAMPLE 3

HCM1 Expression Vectors

A DNA fragment containing the 476-amino acid HCM1 open reading frame wasamplified by polymerase chain reaction from the pHG0376 template(Genbank AB007858) using Pfu DNA polymerase and oligonucleotide primersdesigned to introduce an NdeI restriction site at the translation startcodon and a BamHI site 3' of the stop codon. The PCR product wasdigested with NdeI and BamHII and inserted into the T7 RNApolymerase-based expression plasmid pET16b to generate plasmidpET-His-Hcm1. N-terminal deletion mutants of HCM1 were constructed byPCR amplification with mutagenic sense-strand primers that introduced anNdeI restriction site and a methionine codon in lieu of the codons forGly-120 and Ala-151 or an NdeI restriction site at the Met-179 codon.The PCR products were digested with NdeI and BamHI, then inserted intopET16b to yield plasmids pET-His-Hcm1(121-476), pET-His-Hcm1(152-476),and pET-His-Hcm1(179-476). The inserts of each plasmid were sequenced toverify that no unwanted coding changes were introduced duringamplification and cloning.

EXAMPLE 4

Hcm1p Expression and Purification

pET-His-Hcm1 was transformed into Escherichia coli BL21(DE3). A 1-literculture of E. coli BL21(DE3)/pET-His-Hcm1 was grown at 37° C. inLuria-Bertani medium containing 0.1 mg/ml ampicillin until the A₆₀₀reached 0.5. The culture was adjusted to 0.4 mMisopropyl-β-D-thiogalactopyranoside (IPTG) and incubation was continuedat 30° C. for 4 h. Cells were harvested by centrifugation and the pelletwas stored at -80° C. All subsequent procedures were performed at 4° C.Thawed bacteria were resuspended in 50 ml of lysis buffer (50 mM TrisHCl [pH 7.5], 0.15 M NaCl, 10% sucrose). Cell lysis was achieved byaddition of lysozyme and Triton X-100 was to a final concentrations of50 μg/ml and 0.1%, respectively. The lysate was sonicated to reduceviscosity and insoluble material was removed by centrifugation. Thesoluble extract was mixed for 1 h with 2-ml of Ni-NTA-agarose resin thathad been equilibrated with lysis buffer. The suspension was poured intoa column and washed with lysis buffer. The column was eluted step-wisewith IMAC buffer (20 mM Tris HCl [pH 7.9], 0.5 M NaCl, 1 mMphenylmethylsulfonyl fluoride, 10% glycerol) containing 25, 50, 200, and500 mM imidazole. The polypeptide composition of the column fractionswas monitored by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). The recombinant Hcm1p protein was retainedon the column and recovered in the 200 mM imidazole eluate. An aliquotof this fraction (100 μg of protein) was applied to a 4.8-ml 15-30%glycerol gradients containing 0.5 M NaCl in buffer A (50 mM Tris-HCl [pH8.0], 1 mM EDTA, 2 mM DTT, and 0.1% Triton X-100). The gradient werecentrifuged at 50,000 rpm for 15 h at 4° C. in a Beckman SW50 rotor.Fractions (˜0.2 ml) were collected from the bottom of the tube. Proteinconcentration was determined using the BioRad dye reagent with bovineserum albumin as the standard.

EXAMPLE 5

Expression and Purification of N-Terminal Deletion Mutants of Hcm1p

A modified strategy was employed to optimize the expression of theHcm1(121-476)p and Hcm1(152-476)p proteins in soluble form in bacteria.Cultures (1-liter) of E. coli BL21(DE3) bearing the pET-based plasmidswere grown at 37° C. until the A₆₀₀ reached 0.5. The cultures wereadjusted to 2% ethanol and incubation was continued at 17° C. for 24 h.The recombinant Hcm1(121-476)p and Hcm1(152-476)p proteins were purifiedfrom soluble lysates by Ni-agarose chromatography and glycerol gradientsedimentation as described above for wild type Hcm1p. Hcm1(179-476)p wasexpressed in bacteria, but was recovered exclusively in the insolublepellet fraction of the cell lysate.

EXAMPLE 6

Yeast HCM1 Expression Plasmids

NdeI-BamHI restriction fragments containing the wild type HCM1 gene andthe N-terminal deletion mutants were excised from the respectivepET16b-based plasmids and inserted into a customized yeast expressionvector pYX1-His, a derivative of pYX132 (CEN TRP1) in which sixconsecutive histidine codons and a unique Nde1 site are inserted betweenthe Nco1 and BamHI sites of pYX132. (pYX132 was purchased from Novagen).The single copy expression plasmids were named pYX-Hcm1,pYX-Hcm1(121-476), pYX-Hcm1(152-476), and pYX-Hcm1(179-476). NcoI-XhoIfragments containing the wild type HCM1 gene and the N-terminal deletionmutants were excised from the respective pYX-based CEN plasmids andinserted into the yeast expression vector pYX232 (2μ TRPI). In thesevectors, expression of the human methyltransferase is under the controlof the yeast TPI1 promoter.

EXAMPLE 7

Expression Plasmids for Yeast Capping Enzymes

Plasmids p360-CEG1/CET1 (CEN URA3 CET1 CEG1) and p358-CET1/CEG1 (CENTRP1 CET1 CEG1) contain the yeast RNA triphosphatase andguanylyltransferase genes under the control of their natural promoters;the CET1 and CEG1 genes are arrayed head-to-head and transcribeddivergently. Plasmids p360-CET1/CEG1/ABD1 (CEN URA3 CET1 CEG1 ABD1) andp358-CET1/CEG1/ABD1 (CEN TRP1 CET1 CEG1 ABD1) were constructed byinsertion of ABD1 (under the control of its natural promoter) intop360-CEG1/CET1 and p358-CET1/CEG1, respectively; the ABD1 gene wasplaced next to CEG1 in a tail-to-tail orientation.

EXAMPLE 8

Yeast Strains

Yeast strain YBS50 (MATa leu2 ade2 trp1 his3 ura3 can1 ceg1::hisGcet1::LEU2 p360-CET1/CEG1) is deleted at the chromosomal loci encodingRNA triphosphatase and guanylyltransferase. YBS40 (MATa leu2 ade2 trp1his3 ura3 can1 abd1::hisG p360-ABD1) is deleted at the chromosomal locusencoding cap methyltransferase YBS52 (MA Ta leu2 ade2 trp1 his3 ura3can1 ceg1::hisG cet1::LEU2 abd1::KAN p360-CET1/CEG1/ABD1) is deleted atthe chromosomal loci encoding all three components of the yeast cappingapparatus. YBS50, YBS40, and YBS52 were derived by targeted genedisruptions in the diploid strain W303, followed by tetrad dissectionand genotyping of haploid progeny. Gene disruptions were confirmed bySouthern blotting.

EXAMPLE 9

RNA Triphosphatase Activity of Cet1p is Essential for Yeast Cell Growth

The fungal RNA triphosphatase Cet1p is a promising drug target becauseit executes the first step of RNA cap formation via a reaction mechanismthat is completely different from that used by the mammalian RNAtriphosphatase. Before attempting to exploit the distinctions incatalytic mechanism to design approaches to antifungal drug discovery,one needs to ascertain that the RNA triphosphatase activity of Cet1p isessential for yeast cell growth. This was shown to be the case bycharacterizing the effects of single amino acid mutations on Cet1pfunction in vitro and in vivo. Mutations were targeted to conservedresidues within motifs A, B, and C of the metal-dependent RNAtriphosphatase/NTPase family (FIG. 4).

Cet1p residues Glu-305 and Glu-307 (motif A), Arg-454 (motif B), andGlu-492, Glu-494, and Glu-496 (motif C) were replaced individually byalanine. The Ala mutations were introduced into Cet1(201-549)p, which isfully active in vitro and in vivo [11]. The six mutated proteins wereexpressed as His-tagged fusions and purified from soluble lysates byNi-agarose column chromatography in parallel with wild typeCet1(201-549)p (FIG. 6A). The RNA triphosphatase activities of the wildtype and six mutant Cet1(201-549)p proteins were assayed by the releaseof ³² Pi from 2 μM [γ³² P]-labeled poly(A). Specific enzyme activity wasdetermined from the slopes of the protein titration curves in the linearrange of enzyme-dependence (FIG. 6B). The specific activity of the wildtype enzyme (16.5 nmol Pi released per microgram of protein in 15 min)corresponds to turnover number of ˜0.8 s⁻¹. Analysis of RNA triphosphatecleavage by wild type Cet1(201-549)p as a function of RNA substrateconcentration revealed a Km of 1 μM for poly(A) triphosphate termini andVmax of 1 s⁻¹. The specific activities of the alanine mutants,calculated from the data in FIG. 6B and expressed as the percent of thewild type value, were: E305A (0.03%); E307A (0.03%); R454A (0.2%); E492A(1.7%); E494A (0.03%); E496A (0.8%).

Mutant alleles of CET1(201-549) encoding triphosphatase-defectiveenzymes were tested for their function in vivo using the plasmid shuffleassay described by Ho et al. [11]. The wild type and mutants codingsequences were cloned into a CEN TRP1 vector so as to place theCET1(201-549) gene under the control of the natural CET1 promoter. Theplasmids were transformed into the Δcet1 strain YBS20, in which thechromosomal CET1 locus has been deleted and replaced by LEU2. Growth ofYBS20 is contingent of the maintenance of a wild type CET1 allele on aCEN URA3 plasmid. Hence, YBS20 is unable to grow on agar mediumcontaining 0.75 mg/ml of 5-fluoroorotic acid (5-FOA), which selectsagainst the URA3 plasmid, unless it is transformed with a biologicallyactive CET1 allele or a functional homologue from another organism.Growth on 5-FOA was complemented by CET1(201-549), but not by mutantalleles E305A, E307A, R454A, E492A, E494A, or E496A [12]. Thecorrelation of in vitro and in vivo mutational effects indicates thatthe RNA triphosphatase activity of Cet1p is essential for yeast cellgrowth.

EXAMPLE 10

Manganese- and Cobalt-Dependent NTPase Activity of Ceg1p

FIG. 7 illustrates that Ceg1p also catalyzes the near-quantitativerelease of ³² Pi from 1 mM [γ³² P]ATP in the presence of 1 mM manganeseas the divalent cation cofactor. There was no detectable ATP hydrolysisin the absence of a divalent cation and 1 mM magnesium was extremelyfeeble in supporting catalysis. The failure of previous investigators toappreciate the NTPase activity of the capping enzyme isolated from yeastextracts or recombinant Cet1p is most likely attributable to thereliance on magnesium as the divalent cation cofactor.

Divalent cation specificity was tested in reaction mixtures containing 1mM ATP and 2 mM divalent cation. Cobalt was at least as effective asmanganese in activating the ATPase. Calcium, copper, and zinc did notactivate the ATPase. Cofactor titration experiments showed thathydrolysis of 1 mM ATP was optimal at 1 to 3 mM MnCl₂. Cobalt-dependenthydrolysis of 1 mM ATP was optimal at 1 to 5 mM CoCl₂. ATP hydrolysiswas measured in 50 mM Tris buffer from pH 6.0 to pH 9.5. Activity wasoptimal from pH 6.5 to pH 7.0 and declined with increased alkalinity.Activity at pH 9.5 was 25% that at pH 7.0.

EXAMPLE 11

Kinetics of ATP Hydrolysis by Cet1p

The extent of ³² Pi release from [γ³² P]ATP during a 15 min reaction wasproportional to the amount of input Cet1p protein (FIG. 8A). ³² Piaccumulated with time over 30 min; the rate of reaction varied linearlywith Cet1p concentration (FIG. 8B). From a plot of initial rate versusenzyme concentration, a turnover number of 25 s⁻¹ was calculated. Cet1pcatalyzed the quantitative conversion of [α³² P]ATP to [α³² P]ADP. Therate of [α³² P]ADP formation was identical to the rate of ³² Pi releasefrom [γ³² P]ATP assayed in a parallel reaction mixture (FIG. 8C). Noformation of [α³² P]AMP from [α³² P]ATP was detected, even after 20-45min of incubation, by which time all of the nucleotide had beenconverted to ADP. Thus, Cet1p catalyzes the hydrolysis of ATP to ADPplus Pi and is unable to further hydrolyze the ADP reaction product.Kinetic parameters were determined by measuring ATPase activity as afunction of input [γ³² P]ATP concentration (FIG. 8D). From adouble-reciprocal plot of the data, a Km of 2.8 μM ATP was calculatedand a Vmax of 25 s⁻¹ (FIG. 8D). The phosphohydrolase activity of Cet1pwas not restricted to ATP. Cet1p also catalyzed manganese-dependenthydrolysis of [α³² P]GTP to [α³² P]GDP, [α³² P]dCTP to [a³² P]dCDP, and[α³² P]dATP to [α³² P]dADP.

The turnover number of the yeast enzyme in ATP hydrolysis (25 s⁻¹) issimilar to the values reported for the baculovirus (30 s⁻¹) and vacciniavirus (10 s⁻¹) triphosphatases, although the affinity of the yeastenzyme for ATP (Km=2.8 μM) is significantly higher than that of eitherLEF-4 (Km=43 μM) or vaccinia triphosphatase (Km=800 μM) [12].

EXAMPLE 12

ATPase Activity of Cet1(201-549) and Cet1(246-549)

Two N-terminal truncation mutants, Cet1(201-549)p and Cet1(246-549)p,that retained full activity in catalyzing the release of ³² Pi from γ³²P-labeled triphosphate-terminated poly(A) were previously purified andcharacterized [11]. Similarly, the two truncated proteins were as activeas full-length Cet1p in hydrolyzing ATP (FIG. 3A). Both reactions arecatalyzed by the same catalytic site within the carboxyl-terminaldomain. The kinetic parameters determined for ATP hydrolysis byCet1(201-549)p [Km=3.3 μM ATP, Vmax=33 s⁻¹ ] were similar to those offull-length Cet1p. These data suggest that the deleted N-terminal200-amino acid segment does not contribute to nucleotide binding orreaction chemistry. Hence, purified recombinant Cet1(201-540)p can beused to screen in vitro for candidate inhibitors of the first step offungal cap formation based on the effects of exogenous compounds on theATPase activity of Cet1(201-540)p.

EXAMPLE 13

Purification and Characterization of Recombinant Human CapMethyltransferase

The 476-amino acid polypeptide encoded by human cDNA KIAA0398 (Genbankaccession AB007858), named Hcm1p, displays sequence conservationthroughout its length with the 426-amino acid Abd1p protein (FIG. 5).Abd1p contains an 11-amino acid C-terminal extension that has nocounterpart in Hcm1p; the C-terminal decapeptide of Abd1p is dispensablefor cap methyltransferase activity in vitro and in vivo [9]. Hcm1p alsodisplays extensive sequence similarity to the candidate capmethyltransferase from C. elegans (FIG. 5). All six enzymic functionalgroups that have been shown to be essential for cap methylation by Abd1p(denoted by dots above the aligned sequences in FIG. 5) are conserved inHcm1p [8, 9].

The Hcm1p protein was expressed in bacteria fused to shorthistidine-rich amino-terminal leader segment. The His-tag allowed forrapid enrichment of Hcm1p based on the affinity of the tag forimmobilized nickel. The bacterial lysate was applied to Ni-agarose andadsorbed material was step-eluted with increasing concentrations ofimidazole. SDS-PAGE analysis revealed a prominent 56 kDa Coomassieblue-stained species in the 200 mM imidazole eluate (see FIG. 10A). Thispolypeptide was not recovered when lysates of IPTG-induced BL21(DE3)carrying either the pET vector alone or pET plasmids expressing otherHis-tagged gene products were subjected to the same Ni-agarosechromatography procedure.

RNA (guanine-7-)methyltransferase activity of the Ni-agarose Hcm1ppreparation was detected by the conversion of ³² P cap-labeled poly(A)to methylated cap-labeled poly(A) in the presence of AdoMet [7]. Thereaction products were digested to cap dinucleotides with nuclease P1and then analyzed by PEI-cellulose thin layer chromatography, whichresolves the GpppA cap from the methylated cap m7GpppA. The radiolabeledproduct synthesized by Hcm1p (FIG. 9, lane 1) co-migrated with m7GpppAgenerated in a parallel reaction mixture containing purified recombinantvaccinia virus cap methyltransferase (FIG. 9, lane 6). Cap methylationby Hcm1p depended on inclusion of S-adenosylmethionine in the reactionmixture (FIG. 9, lane 2). S-adenosylhomocysteine did not support capmethylation (FIG. 9, lane 3) and was partially inhibitory in thepresence of AdoMet (FIG. 9, lane 4).

Hcm1p was further purified by centrifugation of the Ni-agarose fractionthrough a 15-30% glycerol gradient. Cap methyltransferase activitysedimented as a single peak coincident with the peak of the Hcm1ppolypeptide (FIG. 10A). The apparent sedimentation coefficient of 4 S(relative to markers analyzed in parallel) indicated that therecombinant human cap methyltransferase is a monomer.

Characterization of the enzyme was performed using the peak glycerolgradient fraction of Hcm1p. Methylation of capped poly(A) variedlinearly with input enzyme and was quantitative at saturation (FIG.11B). Hcm1p formed 0.24 fmol of methylated capped ends per fmol ofprotein during a 10 min reaction. MgCl₂ strongly inhibited activity in aconcentration-dependent fashion; methylation was reduced by an order ofmagnitude by 1 mM magnesium. The extent of methylation varied withAdoMet concentration. Half-maximal activity was observed at ˜25 mMAdoMet. The reaction product AdoHcy inhibited cap methylation in aconcentration-dependent manner; cap methylation in the presence of 10 μMAdoMet was reduced by 80% in the presence of 100 μM AdoHcy.

EXAMPLE 14

Effects of N-terminal Deletions on Cap Methyltransferase Activity

His-tagged versions of N-terminal truncated proteins Hcm1(121-476)p,Hcm1(152-476)p, and Hcm1(179-476)p were expressed in E. coli.Hcm1(121-476)p (referred to as Δ120) and Hcm1(152-476)p (Δ151) werepurified from soluble bacterial lysates by Ni-agarose chromatography andglycerol gradient sedimentation (FIGS. 10B and 10C). Hcm1(179-476)p wasinsoluble and therefore not amenable to purification. The Δ120 and Δ151proteins sedimented as discrete peaks of 4 S and 3 S respectively. Bothpreparations displayed cap methyltransferase activity that cosedimentedwith the recombinant proteins. Thus, both truncated enzymes aremonomeric.

SDS-PAGE analysis of the peak glycerol gradient fractions of wild typeHcm1p, Δ120, and Δ151 revealed that the proteins were of comparablepurity and that the truncated versions migrated more rapidly than thefull-sized Hcm1p, as expected (FIG. 11A). The specific activities ofΔ120 and Δ151 in cap methylation were calculated from the slopes of thetitration curves in the linear range of enzyme-dependence (FIG. 11B).Δ120 retained 50% of the activity of full-length Hcm1p. Δ151 was 22% asactive as the wild type enzyme.

EXAMPLE 15

Human Cap Methyltransferase is Functional In Vivo in Yeast

HCM1 and the truncated alleles HCM1(121-476), HCM1(152-476), andHCM1(179-476) were cloned into a yeast 2μ TRP1 plasmid such that theirexpression was under the control of the yeast TPI1 promoter. The HCM1plasmids were introduced into yeast strain YBS10 in which thechromosomal ABD1 locus was deleted. Growth of YBS10 is contingent onmaintenance of an extrachromosomal ABD1 gene on a CEN URA3 plasmid. Trp+transformants were plated on medium containing 5-FOA to select againstthe URA3 ABD1 plasmid. Control cells transformed with a TRP1 ABD1plasmid grew on 5-FOA, whereas cells transformed with the TRP1 vectorwere incapable of growth on 5-FOA (FIG. 5A). The instructive finding wasthat cells bearing the HCM1 or HCM1(121-476) plasmids grew on 5-FOA.Thus, human cap methyltransferase was functional in vivo in lieu of theendogenous yeast enzyme. The more extensively truncated allelesHCM1(152-476) and HCM1(179-476) did not support growth on FOA (FIG.12A).

The full-length and truncated HCM1 alleles were also cloned into CENTRP1 vectors. Expression of the Δ120 protein in single-copy complementedthe abdI deletion, whereas expression of the full-length Hcm1p proteindid not (FIG. 12B). The copy-number dependence of complementation byfull-length HCM1 may be indicative of lower levels of Hcm1p expressionin yeast compared to the Δ120 derivative.

Based on the alignment in FIG. 5, the Δ120 deletion of Hcm1p would beroughly comparable to a deletion of 93-amino acids from the N-terminusof yeast Abd1p, whereas the Δ151 deletion would be analogous to removalof 124-amino acids from Abd1p. Deleting 52 or 109 amino acids from theN-terminus of Abd1p did not affect the ability of the deleted alleles tosupport yeast cell growth [8]. However, more extensive deletions of 120,129, or 142 amino acids from the N-terminus were lethal [9]. TheN-terminal margins of the minimal functional domains of the yeast andhuman cap methyltransferases appear to be quite similar.

EXAMPLE 16

Complete Replacement of the Yeast Capping Apparatus by Mammalian Enzymes

The strategy for drug discovery underpinning the present invention is toidentify compounds that block cell growth contingent on pathogen-encodedcapping activities without affecting the growth of otherwise identicalcells bearing the capping enzymes of the host organism. For thisapproach to be feasible, the capping systems of interest must beinterchangeable in vivo. Thus, yeast strains were constructed in whichthe entire fungal capping apparatus was replaced by mammalian enzymes.

Expression of the mammalian triphosphatase-guanylyltransferase in yeastcan complement the growth of singly deleted Δceg1 or Δcet1 strains [11,17, 18, 20]. The critical next step was to test the ability of themammalian capping enzyme to complement a new Δcet1 Δceg1 double-deletionstrain (YBS50), growth of which depends on maintenance of a CEN URA3CET1 CEG1 plasmid. Control experiments showed that transformation ofYBS50 with a CEN TRP1 CET1 CEG1 plasmid permitted the cells to grow on5-FOA, whereas a CEN TRP1 plasmid containing only CET1 or only CEG1 wasunable to rescue growth on 5-FOA (FIG. 10A). Expression of MCE1 on a CENTRPI plasmid under control of the yeast TPI1 promoter fully complementedthe Δcet1 Δceg1 double-deletion (FIG. 13A).

A new Δcet1 Δceg1 Δabd1 triple-deletion strain (YBS52), was thenconstructed, growth of which is sustained by a CEN URA3 CET1 CEG1 ABD1plasmid. Control plasmid shuffle experiments showed that YBS52 cellstransformed with a CEN TRP1 CET1 CEG1 ABD1 plasmid grew on 5-FOA,whereas plasmids containing only CET1, only CEG1, or only ABD1 did notcomplement growth on 5-FOA. Cotransformation with MCE1 plusHCM1(121-476) complemented the triple-deletion; neither MCE1 alone norHCM1(121-476) alone permitted growth of YBS52 on 5-FOA. FOA-resistantisolates were then streaked on YPD plates at 30° C. Using colony size asa rough estimate of growth, cells containing either MCE1 in place ofCET1 plus CEG1, or HCM1(121-476) in place of ABD1 grew about as well asthe strain containing an all-yeast capping apparatus (FIG. 13B).However, colony size was smaller when all three yeast genes werereplaced by MCE1 plus HCM1(121-476) on CEN plasmids (FIG. 13B). Colonysize was increased when the yeast genes were replaced with CEN MCE1 plus2μ HCM1(121-476), implying that the human methyltransferase was limitingfor growth in single copy in this background.

To better gauge the growth of isogenic yeast cells containing yeastversus mammalian capping enzyme components, their doubling times weremeasured in YPD medium in suspension cultures at 30° C. The growth ratesof cells containing expressing either Mce1p or Hcm1(121-476)p in lieu ofthe yeast enzymes (generation times 1.6 to 1.8 h) were similar to thatof cells with an all-yeast capping system (1.4 h). Cells with anall-mammalian capping system expressed from single copy plasmids grewmore slowly (doubling time 3.3 h), but the defect was suppressed byhigh-copy expression of the human methyltransferase (doubling time 2.2h). Thus, the entire three-component fungal capping system can bereplaced by the two-component mammalian system.

The two yeast strains provide the tools to implement the method of drugdiscovery outlined in this application. The method consists of a seriesof primary and secondary screening steps that are outlined below. Theprocedures described in the following section are applicable to theembodiment of the invention that uses yeast as the target organism in ascreen for molecules that target the fungal capping apparatus. Otherembodiments can be adapted as discussed above.

EXAMPLE 17

Screening for Differential Growth Inhibition Based on the Composition orSource of the Capping Apparatus

The two yeast strains used for screening differential growth inhibitionare the "fungal capping strain" (FCS) and "mammalian capping strain"(MCS), respectively. The primary screen entails plating a suspension offungal capping strain cells on one agar plate and a suspension ofmammalian capping strain cells on another plate. A matrix of testcompounds is applied to the two plates in parallel, e.g., via depositionof a solution of each compound within a small hole created in the agar.The compound solution will diffuse radially from the point ofapplication and, if growth-inhibitory, will result in a gradient ofno-growth or slow-growth emanating radially from the point source.Hence, after the plates are incubated for several days at permissivegrowth temperature (30° C. for the fungal capping strain and mammaliancapping strain yeast strains), a halo of no-growth or slow-growth willbe apparent against a background lawn of yeast cells. Compounds thatinhibit growth of fungal capping strain and mammalian capping strain tothe same extent are presumed to be nonspecific inhibitors of yeastgrowth. Capping-specific inhibitors are those that inhibit growth of oneof the two test strains, but not the other. For example, a compound thatinhibits fungal capping strain but not mammalian capping strain is apresumed to exert its effects via the fungal capping apparatus.

Delineation of which of the three fungal capping enzymes is targeted bya candidate anti-fungal capping strain compound identified in step 1 isaccomplished by a second round of screening for differential growthinhibition using strains which contain a mixture of fungal and mammaliancapping components. Derivatives of the triple-knockout yeast strainYBS52 (Δcet1 Δceg1 Δabd1) have been generated that contain: (i) fungaltriphosphatase and guanylyltransferase plus mammalian capmethyltransferase, (ii) mammalian triphosphatase-guanylyltransferaseplus fungal cap methyltransferase. Compounds that inhibited the fungalcapping strain strain by targeting the fungal triphosphatase orguanylyltransferase will inhibit strain (i), but not strain (ii).Compounds that targeted the fungal methyltransferase will inhibit strain(ii), but not strain (i).

Anti-FCS compounds found during step 2 to target the fungaltriphosphatase and/or guanylyltransferase are subjected to an additionalscreen to gauge which of the fungal enzymes is responsible for selectivegrowth inhibition. For this purpose, a yeast strain was constructed thatcontains fungal triphosphatase and mammalian guanylyltransferase.

EXAMPLE 18

In Vitro Screening for Inhibitors of Fungal RNA Triphosphatase

Cet1p displays robust nucleoside triphosphatase (NTPase) activity in thepresence of manganese or cobalt as the divalent cation cofactor. TheNTPase function of Cet1p is mediated by the same catalytic moieties onthe enzyme that carry out the RNA triphosphatase reaction. Hence, assayof the hydrolysis of NTP by Cet1p offers a much more convenient assaythan RNA triphosphatase for conducting large scale testing of Cet1pinhibitors. This is because NTPs are commercially available (includingradioactively labeled NTPs), whereas the synthesis oftriphosphate-terminated RNA is technically complex.

Detection of ATP hydrolysis in the experiment in FIG. 7 entailed the useof radiolabeled ATP and product analysis by thin layer chromatography.However, the assay is easily adapted to a non-radioactive calorimetricmethod of detection of Pi release from ATP [49]. A colorimetric assay ofCet1p activity is especially conducive to high-throughput screening ofcandidate inhibitors.

The following referenes were cited herein:

1. Shuman, S. (1995) Prog. Nucleic Acid Res. Mol. Biol. 50, 101-129.

2. Schwer, et al., (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 4328-4332.

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - <160> NUMBER OF SEQ ID NOS: 114                                      - - <210> SEQ ID NO 1                                                        <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif I of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 1                                                         - - Lys Thr Asp Gly Leu Arg                                                                  5                                                              - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif III of RNA guany - #lyltransferase.             - - <400> SEQUENCE: 2                                                         - - Thr Leu Leu Asp Gly Glu Leu Val                                                          5                                                              - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IIIa of RNA guan - #ylyltransferase.            - - <400> SEQUENCE: 3                                                         - - Arg Tyr Leu Met Phe Asp Cys Leu Ala Ile As - #n Gly                                      5  - #                 10                                      - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IV of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 4                                                         - - Asp Gly Leu Ile Phe                                                                      5                                                              - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 13                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif V of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 5                                                         - - Leu Leu Lys Trp Leu Pro Glu Gln Glu Asn Th - #r Val Asp                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif VI of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 6                                                         - - Trp Glu Met Leu Arg Phe Arg Asp Asp Lys                                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Schizosaccharomyces pombe                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif I of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 7                                                         - - Lys Ser Asp Gly Ile Arg                                                                  5                                                              - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Schizosaccharomyces pombe                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif III of RNA guany - #lyltransferase              - - <400> SEQUENCE: 8                                                         - - Thr Leu Leu Asp Gly Glu Leu Val                                                          5                                                              - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Schizosaccharomyces pombe                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IIIa of RNA guan - #ylyltransferase             - - <400> SEQUENCE: 9                                                         - - Arg Tyr Leu Val Phe Asp Cys Leu Ala Cys As - #p Gly                                      5  - #                 10                                      - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Schizosaccharomyces pombe                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IV of RNA guanyl - #yltransferase               - - <400> SEQUENCE: 10                                                        - - Asp Gly Leu Ile Phe                                                                      5                                                              - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 13                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Schizosaccharomyces pombe                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif V of RNA guanyly - #ltransferase                - - <400> SEQUENCE: 11                                                        - - Leu Leu Lys Trp Lys Pro Lys Glu Met Asn Th - #r Ile Asp                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Schizosaccharomyces pombe                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif VI of RNA guanyl - #yltransferase               - - <400> SEQUENCE: 12                                                        - - Trp Arg Phe Leu Arg Phe Arg Asp Asp Lys                                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Candida albicans                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif I of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 13                                                        - - Lys Thr Asp Gly Leu Arg                                                                  5                                                              - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Candida albicans                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif III of RNA guany - #lyltransferase.             - - <400> SEQUENCE: 14                                                        - - Thr Leu Leu Asp Gly Glu Leu Val                                                          5                                                              - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Candida albicans                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IIIa of RNA guan - #ylyltransferase.            - - <400> SEQUENCE: 15                                                        - - Arg Tyr Val Ile Phe Asp Ala Leu Ala Ile Hi - #s Gly                                      5  - #                 10                                      - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Candida albicans                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IV of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 16                                                        - - Asp Gly Leu Ile Tyr                                                                      5                                                              - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 13                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Candida albicans                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif V of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 17                                                        - - Leu Leu Lys Trp Lys Pro Ala Glu Glu Asn Th - #r Val Asp                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Candida albicans                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif VI of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 18                                                        - - Trp Glu Met Leu Arg Phe Arg Asn Asp Lys                                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 19                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Chlorella virus PBCV-1                                        <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif I of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 19                                                        - - Lys Thr Asp Gly Ile Arg                                                                  5                                                              - -  - - <210> SEQ ID NO 20                                                  <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Chlorella virus PBCV-1                                        <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif III of RNA guany - #lyltransferase.             - - <400> SEQUENCE: 20                                                        - - Ser Ile Phe Asp Gly Glu Leu Cys                                                          5                                                              - -  - - <210> SEQ ID NO 21                                                  <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Chlorella virus PBCV-1                                        <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IIIa of RNA guan - #ylyltransferase.            - - <400> SEQUENCE: 21                                                        - - Ala Phe Val Leu Phe Asp Ala Val Val Val Se - #r Gly                                      5  - #                 10                                      - -  - - <210> SEQ ID NO 22                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Chlorella virus PBCV-1                                        <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IV of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 22                                                        - - Asp Gly Leu Ile Ile                                                                      5                                                              - -  - - <210> SEQ ID NO 23                                                  <211> LENGTH: 13                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Chlorella virus PBCV-1                                        <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif V of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 23                                                        - - Leu Phe Lys Leu Lys Pro Gly Thr His His Th - #r Ile Asp                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 24                                                  <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Chlorella virus PBCV-1                                        <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif VI of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 24                                                        - - Trp Lys Tyr Ile Gln Gly Arg Ser Asp Lys                                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 25                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Caenorhabditis elegans                                        <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif I of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 25                                                        - - Lys Ala Asp Gly Met Arg                                                                  5                                                              - -  - - <210> SEQ ID NO 26                                                  <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Caenorhabditis elegans                                        <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif III of RNA guany - #lyltransferase.             - - <400> SEQUENCE: 26                                                        - - Thr Leu Val Asp Thr Glu Val Ile                                                          5                                                              - -  - - <210> SEQ ID NO 27                                                  <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Caenorhabditis elegans                                        <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IIIa of RNA guan - #ylyltransferase.            - - <400> SEQUENCE: 27                                                        - - Arg Met Leu Ile Tyr Asp Ile Met Arg Phe As - #n Ser                                      5  - #                 10                                      - -  - - <210> SEQ ID NO 28                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Caenorhabditis elegans                                        <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IV of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 28                                                        - - Asp Gly Leu Ile Phe                                                                      5                                                              - -  - - <210> SEQ ID NO 29                                                  <211> LENGTH: 13                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Caenorhabditis elegans                                        <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif V of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 29                                                        - - Val Leu Lys Trp Lys Pro Pro Ser His Asn Se - #r Val Asp                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 30                                                  <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Caenorhabditis elegans                                        <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif VI of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 30                                                        - - Trp Lys Phe Met Arg Glu Arg Thr Asp Lys                                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 31                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Mus                                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif I of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 31                                                        - - Lys Ala Asp Gly Thr Arg                                                                  5                                                              - -  - - <210> SEQ ID NO 32                                                  <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Mus                                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif III of RNA guany - #lyltransferase.             - - <400> SEQUENCE: 32                                                        - - Thr Leu Leu Asp Gly Glu Met Ile                                                          5                                                              - -  - - <210> SEQ ID NO 33                                                  <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Mus                                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IIIa of RNA guan - #ylyltransferase.            - - <400> SEQUENCE: 33                                                        - - Arg Tyr Leu Ile Tyr Asp Ile Ile Lys Phe As - #n Ala                                      5  - #                 10                                      - -  - - <210> SEQ ID NO 34                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Mus                                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IV of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 34                                                        - - Asp Gly Leu Ile Phe                                                                      5                                                              - -  - - <210> SEQ ID NO 35                                                  <211> LENGTH: 13                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Mus                                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif V of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 35                                                        - - Ile Leu Lys Trp Lys Pro Pro Ser Leu Asn Se - #r Val Asp                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 36                                                  <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Mus                                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif VI of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 36                                                        - - Trp Val Phe Met Arg Gln Arg Ile Asp Lys                                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 37                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: African swine fever virus                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif I of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 37                                                        - - Lys Ala Asp Gly Ile Arg                                                                  5                                                              - -  - - <210> SEQ ID NO 38                                                  <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: African swine fever virus                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif III of RNA guany - #lyltransferase.             - - <400> SEQUENCE: 38                                                        - - Thr Ile Leu Asp Gly Glu Phe Met                                                          5                                                              - -  - - <210> SEQ ID NO 39                                                  <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: African swine fever virus                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IIIa of RNA guan - #ylyltransferase.            - - <400> SEQUENCE: 39                                                        - - Glu Phe Tyr Gly Phe Asp Val Ile Met Tyr Gl - #u Gly                                      5  - #                 10                                      - -  - - <210> SEQ ID NO 40                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: African swine fever virus                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IV of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 40                                                        - - Asp Gly Ile Ile Leu                                                                      5                                                              - -  - - <210> SEQ ID NO 41                                                  <211> LENGTH: 13                                                              <212> TYPE: PRT                                                               <213> ORGANISM: African swine fever virus                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif V of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 41                                                        - - Thr Phe Lys Trp Lys Pro Thr Trp Asp Asn Th - #r Leu Asp                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 42                                                  <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: African swine fever virus                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif VI of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 42                                                        - - Trp Glu Ile Val Lys Ile Arg Glu Asp Arg                                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 43                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Trypanosoma brucei gambiense                                  <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif I of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 43                                                        - - Lys Ala Asp Gly Leu Arg                                                                  5                                                              - -  - - <210> SEQ ID NO 44                                                  <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Trypanosoma brucei gambiense                                  <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif III of RNA guany - #lyltransferase.             - - <400> SEQUENCE: 44                                                        - - Phe Leu Leu Asp Thr Glu Val Val                                                          5                                                              - -  - - <210> SEQ ID NO 45                                                  <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Trypanosoma brucei gambiense                                  <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IIIa of RNA guan - #ylyltransferase.            - - <400> SEQUENCE: 45                                                        - - Asp Phe Ile Tyr Phe Trp Gly Leu Asp Gly Ar - #g Arg                                      5  - #                 10                                      - -  - - <210> SEQ ID NO 46                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Trypanosoma brucei gambiense                                  <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IV of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 46                                                        - - Asp Gly Leu Ile Phe                                                                      5                                                              - -  - - <210> SEQ ID NO 47                                                  <211> LENGTH: 13                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Trypanosoma brucei gambiense                                  <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif V of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 47                                                        - - Leu Ile Lys Trp Lys Pro Val His Leu Cys Th - #r Val Asp                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 48                                                  <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Trypanosoma brucei gambiense                                  <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif VI of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 48                                                        - - Trp Thr Phe Arg Asn Ala Arg Asn Asp Lys                                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 49                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Crithidia fasciculata                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif I of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 49                                                        - - Lys Val Asp Gly Gln Arg                                                                  5                                                              - -  - - <210> SEQ ID NO 50                                                  <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Crithidia fasciculata                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif III of RNA guany - #lyltransferase.             - - <400> SEQUENCE: 50                                                        - - Trp Met Leu Asp Ala Glu Leu Ser                                                          5                                                              - -  - - <210> SEQ ID NO 51                                                  <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Crithidia fasciculata                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IIIa of RNA guan - #ylyltransferase.            - - <400> SEQUENCE: 51                                                        - - Asp Tyr Val Phe Phe Gly Gly Lys Gln Ala Ly - #s Arg                                      5  - #                 10                                      - -  - - <210> SEQ ID NO 52                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Crithidia fasciculata                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IV of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 52                                                        - - Asp Gly Leu Val Phe                                                                      5                                                              - -  - - <210> SEQ ID NO 53                                                  <211> LENGTH: 13                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Crithidia fasciculata                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif V of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 53                                                        - - Leu Leu Lys Trp Lys Pro Leu Ser Leu Cys Th - #r Ala Asp                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 54                                                  <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Crithidia fasciculata                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif VI of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 54                                                        - - Trp Arg Leu His Arg Leu Arg Ser Asp Lys                                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 55                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: baculovirus                                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif I of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 55                                                        - - Lys Leu Asp Gly Met Arg                                                                  5                                                              - -  - - <210> SEQ ID NO 56                                                  <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: baculovirus                                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif III of RNA guany - #lyltransferase.             - - <400> SEQUENCE: 56                                                        - - Val Ala Phe Gln Cys Glu Val Met                                                          5                                                              - -  - - <210> SEQ ID NO 57                                                  <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: baculovirus                                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IIIa of RNA guan - #ylyltransferase.            - - <400> SEQUENCE: 57                                                        - - Asn Arg Thr Gln Tyr Glu Cys Gly Val Asn Al - #a Ser                                      5  - #                 10                                      - -  - - <210> SEQ ID NO 58                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: AcNPV baculovirus                                             <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IV of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 58                                                        - - Asp Gly Tyr Val Val                                                                      5                                                              - -  - - <210> SEQ ID NO 59                                                  <211> LENGTH: 15                                                              <212> TYPE: PRT                                                               <213> ORGANISM: AcNPV baculovirus                                             <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif V of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 59                                                        - - Tyr Val Lys Tyr Lys Trp Met Pro Thr Thr Gl - #u Leu Glu Tyr Asp                          5  - #                 10 - #                 15               - -  - - <210> SEQ ID NO 60                                                  <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: baculovirus                                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif VI of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 60                                                        - - Ile Asn Val Leu Arg His Arg Arg Asp Arg                                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 61                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: vaccinia virus                                                <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif I of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 61                                                        - - Lys Thr Asp Gly Ile Pro                                                                  5                                                              - -  - - <210> SEQ ID NO 62                                                  <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: vaccinia virus                                                <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif III of RNA guany - #lyltransferase.             - - <400> SEQUENCE: 62                                                        - - Val Val Val Phe Gly Glu Ala Val                                                          5                                                              - -  - - <210> SEQ ID NO 63                                                  <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: vaccinia virus                                                <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IIIa of RNA guan - #ylyltransferase.            - - <400> SEQUENCE: 63                                                        - - Asn Trp Thr Val Tyr Leu Ile Lys Leu Ile Gl - #u Pro                                      5  - #                 10                                      - -  - - <210> SEQ ID NO 64                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: vaccinia virus                                                <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IV of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 64                                                        - - Glu Gly Val Ile Leu                                                                      5                                                              - -  - - <210> SEQ ID NO 65                                                  <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: vaccinia virus                                                <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif V of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 65                                                        - - Asp Phe Lys Ile Lys Lys Glu Asn Thr Ile As - #p                                          5  - #                 10                                      - -  - - <210> SEQ ID NO 66                                                  <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: vaccinia virus                                                <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif VI of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 66                                                        - - Gly Glu Ile Leu Lys Pro Arg Ile Asp Lys                                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 67                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Shope fibroma virus                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif I of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 67                                                        - - Lys Thr Asp Gly Val Gly                                                                  5                                                              - -  - - <210> SEQ ID NO 68                                                  <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Shope fibroma virus                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif III of RNA guany - #lyltransferase.             - - <400> SEQUENCE: 68                                                        - - Val Thr Leu Tyr Gly Glu Ala Val                                                          5                                                              - -  - - <210> SEQ ID NO 69                                                  <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Shope fibroma virus                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IIIa of RNA guan - #ylyltransferase.            - - <400> SEQUENCE: 69                                                        - - Val Trp Gln Ile Tyr Leu Ile Lys Leu Ile Th - #r Pro                                      5  - #                 10                                      - -  - - <210> SEQ ID NO 70                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Shope fibroma virus                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IV of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 70                                                        - - Glu Gly Val Leu Leu                                                                      5                                                              - -  - - <210> SEQ ID NO 71                                                  <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Shope fibroma virus                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif V of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 71                                                        - - Asp Tyr Lys Ile Lys Leu Asp Asn Thr Asp As - #p                                          5  - #                 10                                      - -  - - <210> SEQ ID NO 72                                                  <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Shope fibroma virus                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif VI of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 72                                                        - - Gly Glu Ile Leu Asp Pro Arg Ile Asp Lys                                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 73                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: molluscum contagiosum virus                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif I of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 73                                                        - - Lys Thr Asp Gly Val Pro                                                                  5                                                              - -  - - <210> SEQ ID NO 74                                                  <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: molluscum contagiosum virus                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif III of RNA guany - #lyltransferase.             - - <400> SEQUENCE: 74                                                        - - Val Ala Leu Phe Gly Glu Ala Val                                                          5                                                              - -  - - <210> SEQ ID NO 75                                                  <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: molluscum contagiosum virus                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IIIa of RNA guan - #ylyltransferase.            - - <400> SEQUENCE: 75                                                        - - Gln Leu Thr Val Tyr Leu Ile Lys Leu Met Al - #a Pro                                      5  - #                 10                                      - -  - - <210> SEQ ID NO 76                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: molluscum contagiosum virus                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif IV of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 76                                                        - - Glu Gly Val Val Leu                                                                      5                                                              - -  - - <210> SEQ ID NO 77                                                  <211> LENGTH: 11                                                              <212> TYPE: PRT                                                               <213> ORGANISM: molluscum contagiosum virus                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif V of RNA guanyly - #ltransferase.               - - <400> SEQUENCE: 77                                                        - - Asp Leu Lys Leu Lys Arg Asp Asn Thr Val As - #p                                          5  - #                 10                                      - -  - - <210> SEQ ID NO 78                                                  <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: molluscum contagiosum virus                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif VI of RNA guanyl - #yltransferase.              - - <400> SEQUENCE: 78                                                        - - Gly Arg Leu Leu Arg Pro Arg Leu Ala Lys                                                  5  - #                 10                                      - -  - - <210> SEQ ID NO 79                                                  <211> LENGTH: 357                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      <220> FEATURE:                                                                <222> LOCATION: 43..399                                                       <223> OTHER INFORMATION: Amino acid sequence of - #RNA                              guanylyltransferase.                                                     - - <400> SEQUENCE: 79                                                        - - Phe Pro Gly Ser Gln Pro Val Ser Phe Gln Hi - #s Ser Asp Val Glu                          5  - #                 10 - #                 15               - - Glu Lys Leu Leu Ala His Asp Tyr Tyr Val Cy - #s Glu Lys Thr Asp                          20  - #                25  - #                30               - - Gly Leu Arg Val Leu Met Phe Ile Val Ile As - #n Pro Val Thr Gly                          35  - #                40  - #                45               - - Glu Gln Gly Cys Phe Met Ile Asp Arg Glu As - #n Asn Tyr Tyr Leu                          50  - #                55  - #                60               - - Val Asn Gly Phe Arg Phe Pro Arg Leu Pro Gl - #n Lys Lys Lys Glu                          65  - #                70  - #                75               - - Glu Leu Leu Glu Thr Leu Gln Asp Gly Thr Le - #u Leu Asp Gly Glu                          80  - #                85  - #                90               - - Leu Val Ile Gln Thr Asn Pro Met Thr Lys Le - #u Gln Glu Leu Arg                          95  - #                100 - #                105              - - Tyr Leu Met Phe Asp Cys Leu Ala Ile Asn Gl - #y Arg Cys Leu Thr                          110  - #               115  - #               120              - - Gln Ser Pro Thr Ser Ser Arg Leu Ala His Le - #u Gly Lys Glu Phe                          125  - #               130  - #               135              - - Phe Lys Pro Tyr Phe Asp Leu Arg Ala Ala Ty - #r Pro Asn Arg Cys                          140  - #               145  - #               150              - - Thr Thr Phe Pro Phe Lys Ile Ser Met Lys Hi - #s Met Asp Phe Ser                          155  - #               160  - #               165              - - Tyr Gln Leu Val Lys Val Ala Lys Ser Leu As - #p Lys Leu Pro His                          170  - #               175  - #               180              - - Leu Ser Asp Gly Leu Ile Phe Thr Pro Val Ly - #s Ala Pro Tyr Thr                          185  - #               190  - #               195              - - Ala Gly Gly Lys Asp Ser Leu Leu Leu Lys Tr - #p Lys Pro Glu Gln                          200  - #               205  - #               210              - - Glu Asn Thr Val Asp Phe Lys Leu Ile Leu As - #p Ile Pro Met Val                          215  - #               220  - #               225              - - Glu Asp Pro Ser Leu Pro Lys Asp Asp Arg As - #n Arg Trp Tyr Tyr                          230  - #               235  - #               240              - - Asn Tyr Asp Val Lys Pro Val Phe Ser Leu Ty - #r Val Trp Gln Gly                          245  - #               250  - #               255              - - Gly Ala Asp Val Asn Ser Arg Leu Lys His Ph - #e Asp Gln Pro Phe                          260  - #               265  - #               270              - - Lys Arg Lys Glu Phe Glu Ile Leu Glu Arg Th - #r Tyr Arg Lys Phe                          275  - #               280  - #               285              - - Ala Glu Leu Ser Val Ser Asp Glu Glu Trp Gl - #n Asn Leu Lys Asn                          290  - #               295  - #               300              - - Leu Glu Gln Pro Leu Asn Gly Arg Ile Val Gl - #u Cys Ala Lys Asn                          305  - #               310  - #               315              - - Gln Glu Thr Gly Ala Trp Glu Met Leu Arg Ph - #e Arg Asp Asp Lys                          320  - #               325  - #               330              - - Leu Asn Gly Asn His Thr Ser Val Val Gln Ly - #s Val Leu Glu Ser                          335  - #               340  - #               345              - - Ile Asn Asp Ser Val Ser Leu Glu Asp Leu Gl - #u Glu                                      350  - #               355                                     - -  - - <210> SEQ ID NO 80                                                  <211> LENGTH: 333                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Candida albicans                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Amino acid sequence of - #RNA guanylyltransferas    e.                                                                              - - <400> SEQUENCE: 80                                                        - - Phe Pro Gly Ser Gln Pro Val Ser Phe Glu Ar - #g Arg His Leu Glu                          5  - #                 10 - #                 15               - - Glu Thr Leu Met Gln Lys Asp Tyr Phe Val Cy - #s Glu Lys Thr Asp                          20  - #                25  - #                30               - - Gly Leu Arg Cys Leu Leu Phe Leu Ile Asn As - #p Pro Asp Lys Gly                          35  - #                40  - #                45               - - Glu Gly Val Phe Leu Val Thr Arg Glu Asn As - #p Tyr Tyr Phe Ile                          50  - #                55  - #                60               - - Pro Asn Ile His Phe Pro Leu Ser Val Asn Gl - #u Thr Arg Glu Lys                          65  - #                70  - #                75               - - Pro Thr Tyr His His Gly Thr Leu Leu Asp Gl - #y Glu Leu Val Leu                          80  - #                85  - #                90               - - Glu Asn Arg Asn Val Ser Glu Pro Val Leu Ar - #g Tyr Val Ile Phe                          95  - #                100 - #                105              - - Asp Ala Leu Ala Ile His Gly Lys Cys Ile Il - #e Asp Arg Pro Leu                          110  - #               115  - #               120              - - Pro Lys Arg Leu Gly Tyr Ile Thr Glu Asn Va - #l Met Lys Pro Phe                          125  - #               130  - #               135              - - Asp Asn Phe Lys Lys His Asn Pro Asp Ile Va - #l Asn Ser Pro Glu                          140  - #               145  - #               150              - - Phe Pro Phe Lys Val Gly Phe Lys Thr Met Le - #u Thr Ser Tyr His                          155  - #               160  - #               165              - - Ala Asp Asp Val Leu Ser Lys Met Asp Lys Le - #u Phe His Ala Ser                          170  - #               175  - #               180              - - Asp Gly Leu Ile Tyr Thr Cys Ala Glu Thr Pr - #o Tyr Val Phe Gly                          185  - #               190  - #               195              - - Thr Asp Gln Thr Leu Leu Lys Trp Lys Pro Al - #a Glu Glu Asn Thr                          200  - #               205  - #               210              - - Tyr Asp Phe Gln Leu Glu Phe Val Phe Asn Gl - #u Val Gln Asp Pro                          215  - #               220  - #               225              - - Asp Leu Asp Glu Arg Asp Pro Thr Ser Thr Ty - #r Leu Asp Tyr Asp                          230  - #               235  - #               240              - - Ala Lys Pro Asn Leu Ile Lys Leu Arg Val Tr - #p Gln Gly Ser Asn                          245  - #               250  - #               255              - - Val His Thr Asp Phe Ala Lys Leu Asp Leu Se - #r Asp Asp Asp Trp                          260  - #               265  - #               270              - - Glu Arg Leu Lys Ala Leu Glu Gln Pro Leu Gl - #n Gly Arg Ile Ala                          275  - #               280  - #               285              - - Glu Cys Arg Gln Ser Thr Thr Lys Lys Gly Ty - #r Trp Glu Met Leu                          290  - #               295  - #               300              - - Arg Phe Arg Asn Asp Lys Ser Asn Gly Asn Hi - #s Ile Ser Val Val                          305  - #               310  - #               315              - - Glu Lys Ile Leu Val Ser Ile Lys Asp Gly Va - #l Lys Glu Lys Glu                          320  - #               325  - #               330              - - Val Ile Glu                                                               - -  - - <210> SEQ ID NO 81                                                  <211> LENGTH: 318                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Schizosaccharomyces pombe                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Amino acid sequence of - #RNA guanylyltransferas    e.                                                                              - - <400> SEQUENCE: 81                                                        - - Phe Pro Gly Ser Gln Glu Val Ser Phe Ser Ly - #s Lys His Leu Gln                          5  - #                 10 - #                 15               - - Ala Leu Lys Glu Lys Asn Tyr Phe Val Cys Gl - #u Lys Ser Asp Gly                          20  - #                25  - #                30               - - Ile Arg Cys Leu Leu Tyr Met Thr Glu His Pr - #o Arg Tyr Glu Asn                          35  - #                40  - #                45               - - Arg Pro Ser Val Tyr Leu Glu Asp Arg Lys Me - #t Asn Phe Tyr His                          50  - #                55  - #                60               - - Val Glu Lys Ile Phe Tyr Pro Val Glu Asn As - #p Lys Ser Gly Lys                          65  - #                70  - #                75               - - Lys Tyr His Val Asp Thr Leu Leu Asp Gly Gl - #u Leu Val Leu Asp                          80  - #                85  - #                90               - - Ile Tyr Pro Gly Gly Lys Lys Gln Leu Arg Ty - #r Leu Val Phe Asp                          95  - #                100 - #                105              - - Cys Leu Ala Cys Asp Gly Ile Val Tyr Met Se - #r Arg Leu Leu Asp                          110  - #               115  - #               120              - - Lys Arg Leu Gly Ile Phe Ala Lys Ser Ile Gl - #n Lys Pro Leu Asp                          125  - #               130  - #               135              - - Glu Tyr Thr Lys Thr His Met Arg Glu Thr Al - #a Ile Phe Pro Phe                          140  - #               145  - #               150              - - Leu Thr Ser Leu Lys Lys Met Glu Leu Gly Hi - #s Gly Ile Leu Lys                          155  - #               160  - #               165              - - Leu Phe Asn Glu Val Ile Pro Arg Leu Arg Hi - #s Gly Asn Asp Gly                          170  - #               175  - #               180              - - Leu Ile Phe Thr Cys Thr Glu Thr Pro Tyr Va - #l Ser Gly Thr Asp                          185  - #               190  - #               195              - - Gln Ser Leu Leu Lys Trp Lys Pro Lys Glu Me - #t Asn Thr Ile Asp                          200  - #               205  - #               210              - - Phe Met Leu Lys Leu Glu Phe Ala Gln Pro Gl - #u Glu Gly Asp Ile                          215  - #               220  - #               225              - - Asp Tyr Ser Ala Met Pro Glu Phe Gln Leu Gl - #y Val Trp Glu Gly                          230  - #               235  - #               240              - - Arg Asn Met Tyr Ser Phe Phe Ala Phe Met Ty - #r Val Asp Glu Lys                          245  - #               250  - #               255              - - Glu Trp Glu Lys Leu Lys Ser Phe Asn Val Pr - #o Leu Ser Glu Arg                          260  - #               265  - #               270              - - Ile Val Glu Cys Tyr Leu Asp Asp Glu Asn Ar - #g Trp Arg Phe Leu                          275  - #               280  - #               285              - - Arg Phe Arg Asp Asp Lys Arg Asp Ala Asn Hi - #s Ile Ser Thr Val                          290  - #               295  - #               300              - - Lys Ser Val Leu Gln Ser Ile Glu Asp Gly Va - #l Ser Lys Glu Asp                          305  - #               310  - #               315              - - Leu Leu Lys                                                               - -  - - <210> SEQ ID NO 82                                                  <211> LENGTH: 293                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Mus                                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Amino acid sequence of - #RNA guanylyltransferas    e.                                                                              - - <400> SEQUENCE: 82                                                        - - Phe Pro Gly Ala Gln Pro Val Ser Met Asp Ly - #s Gln Asn Ile Arg                          5  - #                 10 - #                 15               - - Leu Leu Glu Gln Lys Pro Tyr Lys Val Ser Tr - #p Lys Ala Asp Gly                          20  - #                25  - #                30               - - Thr Arg Tyr Met Met Leu Ile Asp Gly Thr As - #n Glu Val Phe Met                          35  - #                40  - #                45               - - Ile Asp Arg Asp Asn Ser Val Phe His Val Se - #r Asn Leu Glu Phe                          50  - #                55  - #                60               - - Pro Phe Arg Lys Asp Leu Arg Met His Leu Se - #r Asn Thr Leu Leu                          65  - #                70  - #                75               - - Asp Gly Glu Met Ile Ile Asp Lys Val Asn Gl - #y Gln Ala Val Pro                          80  - #                85  - #                90               - - Arg Tyr Leu Ile Tyr Asp Ile Ile Lys Phe As - #n Ala Gln Pro Val                          95  - #                100 - #                105              - - Gly Asp Cys Asp Phe Asn Ile Arg Leu Gln Cy - #s Ile Glu Arg Glu                          110  - #               115  - #               120              - - Ile Ile Ser Pro Arg His Glu Lys Met Lys Th - #r Gly Leu Ile Asp                          125  - #               130  - #               135              - - Lys Thr Gln Glu Pro Phe Ser Val Arg Arg Ly - #s Gln Phe Phe Asp                          140  - #               145  - #               150              - - Ile Asn Ile Ser Arg Lys Leu Leu Glu Gly As - #n Phe Ala Lys Glu                          155  - #               160  - #               165              - - Val Ser His Glu Met Asp Gly Leu Ile Phe Gl - #n Pro Ile Gly Lys                          170  - #               175  - #               180              - - Tyr Lys Pro Gly Arg Cys Asp Asp Ile Leu Ly - #s Trp Lys Pro Pro                          185  - #               190  - #               195              - - Ser Leu Asn Ser Val Asp Phe Arg Leu Lys Il - #e Thr Arg Met Gly                          200  - #               205  - #               210              - - Gly Glu Gly Leu Leu Pro Gln Asn Val Gly Le - #u Leu Tyr Val Gly                          215  - #               220  - #               225              - - Gly Tyr Glu Arg Pro Phe Ala Gln Ile Lys Va - #l Thr Lys Glu Leu                          230  - #               235  - #               240              - - Lys Gln Tyr Asp Asn Lys Ile Ile Glu Cys Ly - #s Phe Glu Asn Asn                          245  - #               250  - #               255              - - Ser Trp Val Phe Met Arg Gln Arg Ile Asp Ly - #s Ser Phe Pro Asn                          260  - #               265  - #               270              - - Ala Tyr Asn Thr Ala Met Ala Val Cys Asn Se - #r Ile Ser Asn Pro                          275  - #               280  - #               285              - - Val Thr Lys Glu Met Leu Phe Glu                                                          290                                                            - -  - - <210> SEQ ID NO 83                                                  <211> LENGTH: 254                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Caenorhabditis elegans                                        <220> FEATURE:                                                                <223> OTHER INFORMATION: Amino acid sequence of - #RNA guanylyltransferas    e.                                                                              - - <400> SEQUENCE: 83                                                        - - Phe Pro Gly Leu Gln Pro Val Ser Leu Ser Ar - #g Gly Asn Ile Asn                          5  - #                 10 - #                 15               - - Leu Leu Glu Gln Glu Ser Tyr Met Val Ser Tr - #p Lys Ala Asp Gly                          20  - #                25  - #                30               - - Met Arg Tyr Ile Ile Tyr Ile Asn Asp Gly As - #p Val Tyr Ala Phe                          35  - #                40  - #                45               - - Asp Arg Asp Asn Glu Val Phe Glu Ile Glu As - #n Leu Asp Phe Val                          50  - #                55  - #                60               - - Thr Lys Asn Gly Ala Pro Leu Met Glu Thr Le - #u Val Asp Thr Glu                          65  - #                70  - #                75               - - Val Ile Ile Asp Lys Val Glu Ile Asn Gly Al - #a Met Cys Asp Gln                          80  - #                85  - #                90               - - Pro Arg Met Leu Ile Tyr Asp Ile Met Arg Ph - #e Asn Ser Val Asn                          95  - #                100 - #                105              - - Val Met Lys Glu Pro Phe Tyr Lys Arg Phe Gl - #u Ile Ile Lys Thr                          110  - #               115  - #               120              - - Glu Ile Ile Asp Met Arg Thr Ala Ala Phe Ly - #s Thr Gly Arg Leu                          125  - #               130  - #               135              - - Lys His Glu Asn Gln Ile Met Ser Val Arg Ar - #g Lys Asp Phe Tyr                          140  - #               145  - #               150              - - Asp Leu Glu Ala Thr Ala Lys Leu Phe Gly Pr - #o Lys Phe Val Gln                          155  - #               160  - #               165              - - His Val Gly His Pro Glu Trp Ile Gly Tyr Le - #u Phe Val Gln Asn                          170  - #               175  - #               180              - - Leu Ser Asp Pro Phe Gly Thr Met Ala Lys Al - #a Thr Ala Thr Leu                          185  - #               190  - #               195              - - Lys Lys Tyr His Asn Lys Ile Ile Glu Cys Th - #r Leu Leu Val Asp                          200  - #               205  - #               210              - - Asn Gln Gly Arg Pro Lys Glu Trp Lys Phe Me - #t Arg Glu Arg Thr                          215  - #               220  - #               225              - - Asp Lys Ser Leu Pro Asn Gly Leu Arg Thr Al - #a Glu Asn Val Val                          230  - #               235  - #               240              - - Glu Thr Met Val Asn Pro Val Thr Glu Thr Ty - #r Leu Ile Glu                              245  - #               250                                     - -  - - <210> SEQ ID NO 84                                                  <211> LENGTH: 270                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Chlorella virus PBCV-1                                        <220> FEATURE:                                                                <223> OTHER INFORMATION: Amino acid sequence of - #RNA guanylyltransferas    e.                                                                              - - <400> SEQUENCE: 84                                                        - - Leu Pro Gly Pro Asn Pro Val Ser Ile Glu Ar - #g Lys Asp Phe Glu                          5  - #                 10 - #                 15               - - Lys Leu Lys Gln Asn Lys Tyr Val Val Ser Gl - #u Lys Thr Asp Gly                          20  - #                25  - #                30               - - Ile Arg Phe Met Met Phe Phe Thr Arg Val Ph - #e Gly Phe Lys Val                          35  - #                40  - #                45               - - Cys Thr Ile Ile Asp Arg Ala Met Thr Val Ty - #r Leu Leu Pro Phe                          50  - #                55  - #                60               - - Lys Asn Ile Pro Arg Val Leu Phe Gln Gly Se - #r Ile Phe Asp Gly                          65  - #                70  - #                75               - - Glu Leu Cys Val Asp Ile Val Glu Lys Lys Ph - #e Ala Phe Val Leu                          80  - #                85  - #                90               - - Phe Asp Ala Val Val Val Ser Gly Val Thr Va - #l Ser Gln Met Asp                          95  - #                100 - #                105              - - Leu Ala Ser Arg Phe Phe Ala Met Lys Arg Se - #r Leu Lys Glu Phe                          110  - #               115  - #               120              - - Lys Asn Val Pro Glu Asp Pro Ala Ile Leu Ar - #g Tyr Lys Glu Trp                          125  - #               130  - #               135              - - Ile Pro Leu Glu His Pro Thr Ile Ile Lys As - #p His Leu Lys Lys                          140  - #               145  - #               150              - - Ala Asn Ala Ile Tyr His Thr Asp Gly Leu Il - #e Ile Met Ser Val                          155  - #               160  - #               165              - - Asp Glu Pro Val Ile Tyr Gly Arg Asn Phe As - #n Leu Phe Lys Leu                          170  - #               175  - #               180              - - Lys Pro Gly Thr His His Thr Ile Asp Phe Il - #e Ile Met Ser Glu                          185  - #               190  - #               195              - - Asp Gly Thr Ile Gly Ile Phe Asp Pro Asn Le - #u Arg Lys Asn Val                          200  - #               205  - #               210              - - Pro Val Gly Lys Leu Asp Gly Tyr Tyr Asn Ly - #s Gly Ser Ile Val                          215  - #               220  - #               225              - - Glu Cys Gly Phe Ala Asp Gly Thr Trp Lys Ty - #r Ile Gln Gly Arg                          230  - #               235  - #               240              - - Ser Asp Lys Asn Gln Ala Asn Asp Arg Leu Th - #r Tyr Glu Lys Thr                          245  - #               250  - #               255              - - Leu Leu Asn Ile Glu Glu Asn Ile Thr Ile As - #p Glu Leu Leu Asp                          260  - #               265  - #               270              - -  - - <210> SEQ ID NO 85                                                  <211> LENGTH: 168                                                             <212> TYPE: PRT                                                               <213> ORGANISM: baculovirus                                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Amino acid sequence of - #RNA baculovirus                  phosphatase BVP.                                                         - - <400> SEQUENCE: 85                                                        - - Met Phe Pro Ala Arg Trp His Asn Tyr Leu Gl - #n Cys Gly Gln Val                          5  - #                 10 - #                 15               - - Ile Lys Asp Ser Asn Leu Ile Cys Phe Lys Th - #r Pro Leu Arg Pro                          20  - #                25  - #                30               - - Glu Leu Phe Ala Tyr Val Thr Ser Glu Glu As - #p Val Trp Thr Ala                          35  - #                40  - #                45               - - Glu Gln Ile Val Lys Gln Asn Pro Ser Ile Gl - #y Ala Ile Ile Asp                          50  - #                55  - #                60               - - Leu Thr Asn Thr Ser Lys Tyr Tyr Asp Gly Va - #l His Phe Leu Arg                          65  - #                70  - #                75               - - Ala Gly Leu Leu Tyr Lys Lys Ile Gln Val Pr - #o Gly Gln Thr Leu                          80  - #                85  - #                90               - - Pro Pro Glu Ser Ile Val Gln Glu Phe Ile As - #p Thr Val Lys Glu                          95  - #                100 - #                105              - - Phe Thr Glu Lys Cys Pro Gly Met Leu Val Gl - #y Val His Cys Thr                          110  - #               115  - #               120              - - His Gly Ile Asn Arg Thr Gly Tyr Met Val Cy - #s Arg Tyr Leu Met                          125  - #               130  - #               135              - - His Thr Leu Gly Ile Ala Pro Gln Glu Ala Il - #e Asp Arg Phe Glu                          140  - #               145  - #               150              - - Lys Ala Arg Gly His Lys Ile Glu Arg Gln As - #n Tyr Val Gln Asp                          155  - #               160  - #               165              - - Leu Leu Ile                                                               - -  - - <210> SEQ ID NO 86                                                  <211> LENGTH: 175                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Mus                                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Amino acid sequence of - #N-terminal RNA                   triphosphatase domain of the capping - # enzyme.                         - - <400> SEQUENCE: 86                                                        - - Met Ala Tyr Asn Lys Ile Pro Pro Arg Trp Le - #u Asn Cys Pro Arg                          5  - #                 10 - #                 15               - - Arg Gly Gln Pro Val Ala Gly Arg Phe Leu Pr - #o Leu Lys Thr Met                          20  - #                25  - #                30               - - Leu Gly Pro Arg Tyr Asp Ser Gln Val Ala Gl - #u Glu Asn Arg Phe                          35  - #                40  - #                45               - - His Pro Ser Met Leu Ser Asn Tyr Leu Lys Se - #r Leu Lys Val Lys                          50  - #                55  - #                60               - - Met Ser Leu Leu Val Asp Leu Thr Asn Thr Se - #r Arg Phe Tyr Asp                          65  - #                70  - #                75               - - Arg Asn Asp Ile Glu Lys Glu Gly Ile Lys Ty - #r Ile Lys Leu Gln                          80  - #                85  - #                90               - - Cys Lys Gly His Gly Glu Cys Pro Thr Thr Gl - #u Asn Thr Glu Thr                          95  - #                100 - #                105              - - Phe Ile Arg Leu Cys Glu Arg Phe Asn Glu Ar - #g Ser Pro Pro Glu                          110  - #               115  - #               120              - - Leu Ile Gly Val His Cys Thr His Cys Phe As - #n Arg Thr Gly Phe                          125  - #               130  - #               135              - - Leu Ile Cys Ala Phe Leu Val Glu Lys Met As - #p Trp Ser Ile Glu                          140  - #               145  - #               150              - - Ala Ala Val Ala Thr Phe Ala Gln Ala Arg Pr - #o Pro Gly Ile Tyr                          155  - #               160  - #               165              - - Lys Gly Asp Tyr Leu Lys Glu Leu Phe Arg                                                  170  - #               175                                     - -  - - <210> SEQ ID NO 87                                                  <211> LENGTH: 173                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Candida albicans                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Amino acid sequence of - #N-terminal RNA                   triphosphatase domain of the capping - # enzyme.                         - - <400> SEQUENCE: 87                                                        - - Met Gly Leu Pro Asp Arg Trp Leu His Cys Pr - #o Lys Thr Gly Thr                          5  - #                 10 - #                 15               - - Leu Ile Asn Asn Leu Phe Phe Pro Phe Lys Th - #r Pro Leu Cys Lys                          20  - #                25  - #                30               - - Met Tyr Asp Asn Gln Ile Ala Glu Arg Arg Ty - #r Gln Arg His Pro                          35  - #                40  - #                45               - - Ala Glu Val Phe Ser His Pro His Leu His Gl - #y Lys Lys Ile Gly                          50  - #                55  - #                60               - - Leu Trp Ile Asp Leu Thr Asn Thr Asp Arg Ty - #r Tyr Phe Arg Glu                          65  - #                70  - #                75               - - Glu Val Thr Glu His Glu Cys Ile Tyr His Ly - #s Met Lys Met Ala                          80  - #                85  - #                90               - - Gly Arg Gly Val Ser Pro Thr Gln Glu Asp Th - #r Asp Asn Phe Ile                          95  - #                100 - #                105              - - Lys Leu Val Gln Glu Phe His Lys Lys Tyr Pr - #o Asp Arg Val Val                          110  - #               115  - #               120              - - Gly Val His Cys Thr His Gly Phe Asn Arg Th - #r Gly Phe Leu Ile                          125  - #               130  - #               135              - - Ala Ala Tyr Leu Phe Gln Val Glu Glu Tyr Gl - #y Leu Asp Ala Ala                          140  - #               145  - #               150              - - Ile Gly Glu Phe Ala Glu Asn Arg Gln Lys Gl - #y Ile Tyr Lys Gln                          155  - #               160  - #               165              - - Asp Tyr Ile Asp Asp Leu Phe Ala                                                          170                                                            - -  - - <210> SEQ ID NO 88                                                  <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif A of RNA triphos - #phatase.                    - - <400> SEQUENCE: 88                                                        - - Ser Phe Ile Glu Leu Glu Met Lys Phe                                                      5                                                              - -  - - <210> SEQ ID NO 89                                                  <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif B of RNA triphos - #phatase.                    - - <400> SEQUENCE: 89                                                        - - Ile Ser Glu Arg Thr Lys Asp Arg Val Ser Ty - #r Ile His Asn                              5  - #                 10                                      - -  - - <210> SEQ ID NO 90                                                  <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif C of RNA triphos - #phatase.                    - - <400> SEQUENCE: 90                                                        - - Thr His Glu Val Glu Leu Glu                                                              5                                                              - -  - - <210> SEQ ID NO 91                                                  <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: vaccinia virus                                                <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif A of RNA triphos - #phatase.                    - - <400> SEQUENCE: 91                                                        - - Ile Asn Asn Glu Leu Glu Leu Val Phe                                                      5                                                              - -  - - <210> SEQ ID NO 92                                                  <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: vaccinia virus                                                <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif B of RNA triphos - #phatase.                    - - <400> SEQUENCE: 92                                                        - - Val Lys Ile Arg Thr Lys Ile Pro Leu Ser Ly - #s Val His Gly                              5  - #                 10                                      - -  - - <210> SEQ ID NO 93                                                  <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: vaccinia virus                                                <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif C of RNA triphos - #phatase.                    - - <400> SEQUENCE: 93                                                        - - Ser Leu Glu Ile Glu Phe Thr                                                              5                                                              - -  - - <210> SEQ ID NO 94                                                  <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Shope fibroma virus                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif A of RNA triphos - #phatase.                    - - <400> SEQUENCE: 94                                                        - - Met Asn His Glu Val Glu Leu Thr Phe                                                      5                                                              - -  - - <210> SEQ ID NO 95                                                  <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Shope fibroma virus                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif B of RNA triphos - #phatase.                    - - <400> SEQUENCE: 95                                                        - - Val Lys Ile Arg Asn Arg Ile Asn Leu Ser Ly - #s Ile His Gly                              5  - #                 10                                      - -  - - <210> SEQ ID NO 96                                                  <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Shope fibroma virus                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif C of RNA triphos - #phatase.                    - - <400> SEQUENCE: 96                                                        - - Ser Leu Glu Phe Glu Ile Ile                                                              5                                                              - -  - - <210> SEQ ID NO 97                                                  <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: molluscum contagiosum virus                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif A of RNA triphos - #phatase.                    - - <400> SEQUENCE: 97                                                        - - Val His His Glu Val Glu Leu Ile Phe                                                      5                                                              - -  - - <210> SEQ ID NO 98                                                  <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: molluscum contagiosum virus                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif B of RNA triphos - #phatase.                    - - <400> SEQUENCE: 98                                                        - - Val Lys Leu Arg Thr Arg Leu Pro Leu Ala Th - #r Val His Gly                              5  - #                 10                                      - -  - - <210> SEQ ID NO 99                                                  <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: molluscum contagiosum virus                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif C of RNA triphos - #phatase.                    - - <400> SEQUENCE: 99                                                        - - Thr Leu Glu Phe Glu Val Leu                                                              5                                                              - -  - - <210> SEQ ID NO 100                                                 <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: African swine fever virus                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif A of RNA triphos - #phatase.                    - - <400> SEQUENCE: 100                                                       - - Ser Thr Ile Glu Leu Glu Ile Arg Phe                                                      5                                                              - -  - - <210> SEQ ID NO 101                                                 <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: African swine fever virus                                     <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif B of RNA triphos - #phatase.                    - - <400> SEQUENCE: 101                                                       - - Asn His Cys Arg Glu Lys Ile Leu Pro Ser Gl - #u Asn Leu Tyr                              5  - #                 10                                      - -  - - <210> SEQ ID NO 102                                                 <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: molluscum contagiosum virus                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: African swine fever virus - #.                        - - <400> SEQUENCE: 102                                                       - - Leu Tyr Glu Ile Glu Ile Glu                                                              5                                                              - -  - - <210> SEQ ID NO 103                                                 <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: baculovirus                                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif A of RNA triphos - #phatase.                    - - <400> SEQUENCE: 103                                                       - - Phe Val Ile Glu Lys Glu Ile Ser Tyr                                                      5                                                              - -  - - <210> SEQ ID NO 104                                                 <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: baculovirus                                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif B of RNA triphos - #phatase.                    - - <400> SEQUENCE: 104                                                       - - Asn Gly Phe Arg Thr Arg Ile Pro Ile Gln Se - #r Ala Cys Asn                              5  - #                 10                                      - -  - - <210> SEQ ID NO 105                                                 <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: baculovirus                                                   <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif C of RNA triphos - #phatase.                    - - <400> SEQUENCE: 105                                                       - - Arg Leu Glu Tyr Glu Phe Asp                                                              5                                                              - -  - - <210> SEQ ID NO 106                                                 <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif A of RNA triphos - #phatase.                    - - <400> SEQUENCE: 106                                                       - - Ser His Ile Glu Ile Glu Met Lys Phe                                                      5                                                              - -  - - <210> SEQ ID NO 107                                                 <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif B of RNA triphos - #phatase.                    - - <400> SEQUENCE: 107                                                       - - Ile Leu Gln Arg Thr Lys Ser Arg Ser Thr Ty - #r Thr Phe Asn                              5  - #                 10                                      - -  - - <210> SEQ ID NO 108                                                 <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif C of RNA triphos - #phatase.                    - - <400> SEQUENCE: 108                                                       - - Ser His Glu Val Glu Val Glu                                                              5                                                              - -  - - <210> SEQ ID NO 109                                                 <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Candida albicans                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif A of RNA triphos - #phatase.                    - - <400> SEQUENCE: 109                                                       - - Arg Asn Val Glu Leu Glu Leu Lys Phe                                                      5                                                              - -  - - <210> SEQ ID NO 110                                                 <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Candida albicans                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif B of RNA triphos - #phatase.                    - - <400> SEQUENCE: 110                                                       - - Glu Met Val Arg Glu Lys Lys Arg Ile Ser Ty - #r Thr His Pro                              5  - #                 10                                      - -  - - <210> SEQ ID NO 111                                                 <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Candida albicans                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Motif C of RNA triphos - #phatase.                    - - <400> SEQUENCE: 111                                                       - - Lys Tyr Glu Val Glu Leu Glu                                                              5                                                              - -  - - <210> SEQ ID NO 112                                                 <211> LENGTH: 436                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Amino acid sequence of - #yeast cap                        methyltransferase.                                                       - - <400> SEQUENCE: 112                                                       - - Met Ser Thr Lys Pro Glu Lys Pro Ile Trp Me - #t Ser Gln Glu Asp                          5  - #                 10 - #                 15               - - Tyr Asp Arg Gln Tyr Gly Ser Ile Thr Gly As - #p Glu Ser Ser Thr                          20  - #                25  - #                30               - - Val Ser Lys Lys Asp Ser Lys Val Thr Ala As - #n Ala Pro Gly Asp                          35  - #                40  - #                45               - - Gly Asn Gly Ser Leu Pro Val Leu Gln Ser Se - #r Ser Ile Leu Thr                          50  - #                55  - #                60               - - Ser Lys Val Ser Asp Leu Pro Ile Glu Ala Gl - #u Ser Gly Phe Lys                          65  - #                70  - #                75               - - Ile Gln Lys Arg Arg His Glu Arg Tyr Asp Gl - #n Glu Glu Arg Leu                          80  - #                85  - #                90               - - Arg Lys Gln Arg Ala Gln Lys Leu Arg Glu Gl - #u Gln Leu Asp Arg                          95  - #                100 - #                105              - - His Glu Ile Glu Met Thr Ala Asn Arg Ser Il - #e Asn Val Asp Gln                          110  - #               115  - #               120              - - Ile Val Arg Glu His Tyr Asn Glu Arg Thr Il - #e Ile Ala Asn Arg                          125  - #               130  - #               135              - - Ala Lys Arg Asn Leu Ser Pro Ile Ile Lys Le - #u Arg Asn Phe Asn                          140  - #               145  - #               150              - - Asn Ala Ile Lys Tyr Met Leu Ile Asp Lys Ty - #r Thr Lys Pro Gly                          155  - #               160  - #               165              - - Asp Val Val Leu Glu Leu Gly Cys Gly Lys Gl - #y Gly Asp Leu Arg                          170  - #               175  - #               180              - - Lys Tyr Gly Ala Ala Gly Ile Ser Gln Phe Il - #e Gly Ile Asp Ile                          185  - #               190  - #               195              - - Ser Asn Ala Ser Ile Gln Glu Ala His Lys Ar - #g Tyr Arg Ser Met                          200  - #               205  - #               210              - - Arg Asn Leu Asp Tyr Gln Val Val Leu Ile Th - #r Gly Asp Cys Phe                          215  - #               220  - #               225              - - Gly Glu Ser Leu Gly Val Ala Val Glu Pro Ph - #e Pro Asp Cys Arg                          230  - #               235  - #               240              - - Phe Pro Cys Asp Ile Val Ser Thr Gln Phe Cy - #s Leu His Tyr Ala                          245  - #               250  - #               255              - - Phe Glu Thr Glu Glu Lys Ala Arg Arg Ala Le - #u Leu Asn Val Ala                          260  - #               265  - #               270              - - Lys Ser Leu Lys Ile Gly Gly His Phe Phe Gl - #y Thr Ile Pro Asp                          275  - #               280  - #               285              - - Ser Glu Phe Ile Arg Tyr Lys Leu Asn Lys Ph - #e Pro Lys Glu Val                          290  - #               295  - #               300              - - Glu Lys Pro Ser Trp Gly Asn Ser Ile Tyr Ly - #s Val Thr Phe Glu                          305  - #               310  - #               315              - - Asn Asn Ser Tyr Gln Lys Asn Asp Tyr Glu Ph - #e Thr Ser Pro Tyr                          320  - #               325  - #               330              - - Gly Met Tyr Thr Tyr Trp Leu Glu Ala Asp Al - #a Ile Asp Asn Val                          335  - #               340  - #               345              - - Pro Glu Tyr Val Val Pro Phe Glu Thr Leu Ar - #g Ser Leu Ala Asp                          350  - #               355  - #               360              - - Glu Tyr Gly Leu Glu Leu Val Ser Gln Met Pr - #o Phe Asn Lys Phe                          365  - #               370  - #               375              - - Phe Val Gln Glu Ile Pro Lys Trp Ile Glu Ar - #g Phe Ser Pro Lys                          380  - #               385  - #               390              - - Met Arg Glu Gly Leu Gln Arg Ser Asp Gly Ar - #g Tyr Gly Val Glu                          395  - #               400  - #               405              - - Gly Asp Glu Lys Glu Ala Ala Ser Tyr Phe Ty - #r Thr Met Phe Ala                          410  - #               415  - #               420              - - Arg Arg Lys Val Lys Gln Tyr Ile Glu Pro Gl - #u Ser Val Lys Pro                          425  - #               430  - #               435              - - Asn                                                                       - -  - - <210> SEQ ID NO 113                                                 <211> LENGTH: 373                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Candida albicans                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Predicted sequence of cap - # methyltransferase;           Genbank accession Z81038.                                                - - <400> SEQUENCE: 113                                                       - - Met Met Lys Glu Val Leu Asp Ala Phe Arg Ly - #s Ser Gly Glu Ala                          5  - #                 10 - #                 15               - - Glu Gly Phe Gly His Asn Lys Met Ser Ser Se - #r Glu Val Ala Ser                          20  - #                25  - #                30               - - His Tyr Asn Lys Val Leu Gln Val Gly Ile Gl - #u Gly Arg Lys Glu                          35  - #                40  - #                45               - - Ser Arg Ile Phe Phe Met Arg Asn Met Asn As - #n Trp Val Lys Ser                          50  - #                55  - #                60               - - Gln Leu Ile Asn Asp Ala Lys Gln Arg Val As - #n Asp Asn Gly Val                          65  - #                70  - #                75               - - Asn Asn Pro Arg Val Leu Asp Leu Ala Cys Gl - #y Lys Gly Gly Asp                          80  - #                85  - #                90               - - Leu Lys Lys Trp Asp Ile Ala Gly Ala Lys As - #p Val Val Met Ala                          95  - #                100 - #                105              - - Asp Val Ala Asp Val Ser Ile Gln Gln Ala Gl - #u Glu Arg Tyr Lys                          110  - #               115  - #               120              - - Gln Met Phe Gly Tyr Lys Lys Asn Asn Ile Ph - #e Thr Val Gln Phe                          125  - #               130  - #               135              - - Ile Val Ala Asp Cys Thr Lys Glu Asn Leu Gl - #u Asp Arg Ile Glu                          140  - #               145  - #               150              - - Asn Lys Asp Pro Phe Asp Leu Val Ser Cys Gl - #n Phe Ala Leu His                          155  - #               160  - #               165              - - Tyr Ser Phe Val Asp Glu Ala Ser Ala Arg Il - #e Phe Leu Lys Asn                          170  - #               175  - #               180              - - Ala Val Gly Met Leu Lys Pro Gly Gly Val Ph - #e Ile Gly Thr Leu                          185  - #               190  - #               195              - - Pro Asp Ala Asp Arg Ile Val Trp Ser Met Ar - #g Asn Gly Glu Asn                          200  - #               205  - #               210              - - Gly Gln Phe Ala Asn Glu Val Cys Lys Ile Th - #r Tyr Glu Asn Val                          215  - #               220  - #               225              - - Glu Glu Leu Ala Glu Gly Lys Val Pro Leu Ph - #e Gly Ala Lys Phe                          230  - #               235  - #               240              - - His Phe Ser Leu Asp Glu Gln Val Asn Cys Pr - #o Glu Phe Leu Ala                          245  - #               250  - #               255              - - Tyr Phe Pro Leu Val Lys His Leu Leu Glu Gl - #u Leu Asp Met Glu                          260  - #               265  - #               270              - - Leu Leu Phe Val His Asn Phe Ala Glu Ala Il - #e Asn Lys Trp Leu                          275  - #               280  - #               285              - - Glu Pro Gly Arg Arg Leu Leu Glu Ser Met Th - #r Gly Leu Glu Thr                          290  - #               295  - #               300              - - Tyr Pro Asn Glu Lys Leu Ser Gly Lys Ser As - #p Asp Glu Tyr Leu                          305  - #               310  - #               315              - - Glu Ala Lys Ala Lys Leu Asp Ala Phe Pro Gl - #u Asp Glu Arg Ile                          320  - #               325  - #               330              - - Lys Thr Met Gly Thr Leu Ser Lys Ser Glu Tr - #p Glu Ala Ile Cys                          335  - #               340  - #               345              - - Met Tyr Leu Val Phe Gly Phe Arg Lys Lys Ly - #s Ser Glu Ala Glu                          350  - #               355  - #               360              - - Lys Thr Glu Glu Glu Pro Ala Thr Thr Lys Pr - #o Val Ala                                  365  - #               370                                     - -  - - <210> SEQ ID NO 114                                                 <211> LENGTH: 476                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <223> OTHER INFORMATION: Amino acid sequence of - #human cap                        methyltransferase.                                                       - - <400> SEQUENCE: 114                                                       - - Met Ala Asn Ser Ala Lys Ala Glu Glu Tyr Gl - #u Lys Met Ser Leu                          5  - #                 10 - #                 15               - - Glu Ala Gln Ala Lys Ala Ser Val Asn Ser Gl - #u Thr Glu Ser Ser                          20  - #                25  - #                30               - - Phe Asn Ile Asn Glu Asn Thr Thr Ala Ser Gl - #y Thr Gly Leu Ser                          35  - #                40  - #                45               - - Glu Lys Thr Ser Val Cys Arg Gln Val Asp Il - #e Ala Arg Lys Arg                          50  - #                55  - #                60               - - Lys Glu Phe Glu Asp Asp Leu Val Lys Glu Se - #r Ser Ser Cys Gly                          65  - #                70  - #                75               - - Lys Asp Thr Pro Ser Lys Lys Arg Lys Leu As - #p Pro Glu Ile Val                          80  - #                85  - #                90               - - Pro Glu Glu Lys Asp Cys Gly Asp Ala Glu Gl - #y Asn Ser Lys Lys                          95  - #                100 - #                105              - - Arg Lys Arg Glu Thr Glu Asp Val Pro Lys As - #p Lys Ser Ser Thr                          110  - #               115  - #               120              - - Gly Asp Gly Thr Gly Asn Lys Arg Lys Ile Al - #a Leu Glu Asp Val                          125  - #               130  - #               135              - - Pro Glu Lys Gln Lys Asn Leu Glu Glu Gly Hi - #s Ser Ser Thr Val                          140  - #               145  - #               150              - - Ala Ala His Tyr Asn Glu Leu Gln Glu Val Gl - #y Leu Glu Lys Arg                          155  - #               160  - #               165              - - Ser Gln Ser Arg Ile Phe Tyr Leu Arg Asn Ph - #e Asn Asn Trp Met                          170  - #               175  - #               180              - - Lys Ser Val Leu Ile Gly Glu Phe Leu Glu Ly - #s Val Arg Gln Lys                          185  - #               190  - #               195              - - Lys Lys Arg Asp Ile Thr Val Leu Asp Leu Gl - #y Cys Gly Lys Gly                          200  - #               205  - #               210              - - Gly Asp Leu Leu Lys Trp Lys Lys Gly Arg Il - #e Asn Lys Leu Val                          215  - #               220  - #               225              - - Cys Thr Asp Ile Ala Asp Val Ser Val Lys Gl - #n Cys Gln Gln Arg                          230  - #               235  - #               240              - - Tyr Glu Asp Met Lys Asn Arg Arg Asp Ser Gl - #u Tyr Ile Phe Ser                          245  - #               250  - #               255              - - Ala Glu Phe Ile Thr Ala Asp Ser Ser Lys Gl - #u Leu Leu Ile Asp                          260  - #               265  - #               270              - - Lys Phe Arg Asp Pro Gln Met Cys Phe Asp Il - #e Cys Ser Cys Gln                          275  - #               280  - #               285              - - Phe Val Cys His Tyr Ser Phe Glu Ser Tyr Gl - #u Gln Ala Asp Met                          290  - #               295  - #               300              - - Met Leu Arg Asn Ala Cys Glu Arg Leu Ser Pr - #o Gly Gly Tyr Phe                          305  - #               310  - #               315              - - Ile Gly Thr Thr Pro Asn Ser Phe Glu Leu Il - #e Arg Arg Leu Glu                          320  - #               325  - #               330              - - Ala Ser Glu Thr Glu Ser Phe Gly Asn Glu Il - #e Tyr Thr Val Lys                          335  - #               340  - #               345              - - Phe Gln Lys Lys Gly Asp Tyr Pro Leu Phe Gl - #y Cys Lys Tyr Asp                          350  - #               355  - #               360              - - Phe Asn Leu Glu Gly Val Val Asp Val Pro Gl - #u Phe Leu Val Tyr                          365  - #               370  - #               375              - - Phe Pro Leu Leu Asn Glu Met Ala Lys Lys Ty - #r Asn Met Lys Leu                          380  - #               385  - #               390              - - Val Tyr Lys Lys Thr Phe Leu Glu Phe Tyr Gl - #u Glu Lys Ile Asn                          395  - #               400  - #               405              - - Asn Glu Asn Lys Met Leu Leu Lys Arg Pro Gl - #n Ala Leu Glu Pro                          410  - #               415  - #               420              - - Tyr Pro Ala Asn Glu Ser Ser Lys Leu Val Se - #r Glu Lys Val Asp                          425  - #               430  - #               435              - - Asp Tyr Glu His Ala Ala Lys Tyr Met Lys As - #n Ser Gln Val Arg                          440  - #               445  - #               450              - - Leu Pro Leu Gly Thr Leu Ser Lys Ser Glu Tr - #p Glu Ala Thr Ser                          455  - #               460  - #               465              - - Ile Tyr Leu Val Phe Ala Phe Glu Lys Gln Gl - #n                                          470  - #               475                                   __________________________________________________________________________

What is claimed is:
 1. A method of screening for a compound thatinhibits formation of an organism's 5' mRNA cap structure, comprisingthe steps of:a) replacing a host organism's genes encoding entire 5'mRNA capping functions with genes encoding 5' mRNA capping functionsfrom a first organism, thereby producing a host organism expressing thefirst organism's complete capping apparatus; b) replacing a hostorganism's genes encoding entire 5' mRNA capping functions with genesencoding the 5' mRNA capping functions from a second organism, therebyproducing a host organism expressing the second organism's completecapping apparatus, wherein said first organism and said second organismare not the same; c) treating said host organism expressing the firstorganism's complete capping apparatus and said host organism expressingthe second organism's complete capping apparatus with a test compound;and d) comparing growth inhibition of the host organism expressing thefirst organism's complete capping apparatus with growth inhibition ofthe host organism expressing the second organism's complete cappingapparatus, wherein growth inhibition of only the host organismexpressing the first organism's complete capping apparatus relative tothe host organism expressing the second organism's complete cappingapparatus indicates that the test compound inhibits the 5' mRNA cappingfunctions of the first organism, whereas growth inhibition of only thehost organism expressing the second organism's complete cappingapparatus relative to the host organism expressing the first organism'scomplete capping apparatus indicates that the test compound inhibits the5' mRNA capping functions of the second organism.
 2. The method of claim1, wherein the host organism is selected from the group consisting ofviruses, fungal cells, insect cells, plant cells, and mammalian cells.3. The method of claim 2, wherein when said host organism is a virus,comparison of growth inhibition of the virus expressing the firstorganism's capping apparatus with the virus expressing the secondorganism's capping apparatus is selected from the group consisting ofthe number of viral plaques formed on viral-permissive host cells andthe viral plaque size formed on viral-permissive host cells.
 4. Themethod of claim 2, wherein said fungal cells are Saccharomycescerevisiae.
 5. The method of claim 1, wherein said first organism andsaid second organism are selected from the group consisting of viruses,fungi, protozoa, plants, insects and mammals.
 6. The method of claim 1,wherein said test compound is selected from the group consisting ofchemicals, drugs and proteins.
 7. The method of claim 6, wherein saidprotein is encoded by a DNA expressed in said host cells, wherein saidDNA is operably linked to an inducible promoter.
 8. The method of claim1, wherein said 5' mRNA capping function is selected from the groupconsisting of RNA triphosphatase, RNA guanylyltransferase, and RNA(guanine-N7)-methyltransferase.
 9. A method of screening for a compoundthat inhibits formation of an organism's 5' mRNA cap structure,comprising the steps of:a) replacing genes encoding entire 5' mRNAcapping functions from Saccharomyces cerevisiae with genes encoding 5'mRNA capping functions from a first organism, thereby producing S.cerevisiae cells expressing the first organism's complete cappingapparatus; b) replacing genes encoding entire 5' mRNA capping functionsfrom Saccharomyces cerevisiae with genes encoding the 5' mRNA cappingfunctions from a second organism, thereby producing S. cerevisiae cellsexpressing the second organism's complete capping apparatus, whereinsaid first organism and said second organism are not the same; c)treating S. cerevisiae cells expressing the first organism's completecapping apparatus and S. cerevisiae cells expressing the secondorganism's complete capping apparatus with a test compound; and d)comparing growth inhibition of the S. cerevisiae cells expressing thefirst organism's complete capping apparatus with growth inhibition ofthe S. cerevisiae cells expressing the second organism's completecapping apparatus, wherein growth inhibition of only S. cerevisiae cellsexpressing the first organism's capping apparatus relative to S.cerevisiae cells expressing the second organism's complete cappingapparatus indicates that the test compound inhibits the 5' mRNA cappingfunctions of the first organism, whereas growth inhibition of only S.cerevisiae cells expressing the second organism's capping apparatusrelative to S. cerevisiae cells expressing the first organism's completecapping apparatus indicates that the test compound inhibits the 5' mRNAcapping functions of the second organism.
 10. The method of claim 9,wherein said first organism and said second organism are selected fromthe group consisting of viruses, fungi, protozoa, plants, insects andmammals.
 11. The method of claim 9, wherein said test compound isselected from the group consisting of chemicals, drugs and proteins. 12.The method of claim 11, wherein said protein is encoded by a DNAexpressed in said S. cerevisiae cells, wherein said DNA is operablylinked to an inducible promoter.
 13. The method of claim 9, wherein said5' mRNA capping function is selected from the group consisting of RNAtriphosphatase, RNA guanylyltransferase, and RNA(guanine-N7)-methyltransferase.
 14. A method of screening for a compoundthat inhibits formation of an organism's 5' mRNA cap structure,comprising the steps of:a) replacing genes encoding entire 5' mRNAcapping functions from Saccharomyces cerevisiae with genes encoding 5'mRNA capping functions from a fungal organism, thereby producing S.cerevisiae cells expressing the fungal organism's complete cappingapparatus; b) replacing genes encoding entire 5' mRNA capping functionsfrom Saccharomyces cerevisiae with genes encoding the 5' mRNA cappingfunctions from a mammalian organism, thereby producing S. cerevisiaecells expressing the mammalian organism's complete capping apparatus; c)treating S. cerevisiae cells expressing the fungal organism's completecapping apparatus and S. cerevisiae cells expressing the mammalianorganism's complete capping apparatus with a test compound; and d)comparing growth inhibition of the S. cerevisiae cells expressing thefungal organism's complete capping apparatus with the S. cerevisiaecells expressing the mammalian organism's complete capping apparatus,wherein growth inhibition of only S. cerevisiae cells expressing thefungal organism's complete capping apparatus relative to S. cerevisiaecells expressing the mammalian organism's complete capping apparatusindicates that the test compound inhibits the 5' mRNA capping functionsof the fungal organism, whereas growth inhibition of only S. cerevisiaecells expressing the mammalian organism's complete capping apparatusrelative to S. cerevisiae cells expressing the fungal organism's cappingapparatus indicates that the test compound inhibits the 5' mRNA cappingfunctions of the mammalian organism.
 15. The method of claim 14, whereinsaid test compound is selected from the group consisting of chemicals,drugs and proteins.
 16. The method of claim 15, wherein said protein isencoded by a DNA expressed in said S. cerevisiae cells, wherein said DNAis operably linked to an inducible promoter.
 17. The method of claim 14,wherein said 5' mRNA capping function is selected from the groupconsisting of RNA triphosphatase, RNA guanylyltransferase, and RNA(guanine-N7)-methyltransferase.
 18. A method of screening for a compoundthat inhibits the catalytic activity of fungal RNA 5' triphosphatase,comprising the steps of:a) contacting said fungal RNA 5' triphosphatasewith a nucleoside triphosphate and a divalent cation cofactor in thepresence and absence of a test compound; and b) detecting hydrolysis ofsaid nucleoside triphosphate, wherein a lack of hydrolysis of saidnucleoside triphosphate or a decrease in hydrolysis of said nucleosidetriphosphate indicates inhibition of said fungal RNA 5' triphosphataseby said test compound.
 19. The method of claim 18, wherein said divalentcation cofactor is selected from the group consisting of manganese andcobalt.
 20. The method of claim 18, wherein said detection of hydrolysisis selected from the group consisting of a radioisotope assay and acalorimetric assay.